Probiotic, lactic acid-producing bacteria and uses thereof

ABSTRACT

The present invention discloses compositions and methodologies for the utilization of probiotic organisms in therapeutic compositions. More specifically, the present invention relates to the utilization of one or more species or strains of lactic acid-producing bacteria, preferably strains of  Bacillus coagulans , for the control of gastrointestinal tract pathogens, including antibiotic-resistant gastrointestinal tract pathogens, and their associated diseases by both a reduction in the rate of colonization and the severity of the deleterious physiological effects of the colonization of the antibiotic-resistant pathogen. In addition, the present invention relates to the utilization of therapeutic compounds comprised of lactic acid-producing bacteria and anti-microbial agents such as antibiotics, anti-fungal compounds, anti-yeast compounds, or anti-viral compounds. The present invention also discloses methodologies for: (i) the selective breeding and isolation of probiotic, lactic acid-producing bacterial strains which possess resistance or markedly decreased sensitivity to anti-microbial agents (e.g., antibiotics, anti-fungal agents, anti-yeast agents, and anti-viral agents); and (ii) treating or preventing bacteria-mediated infections of the gastrointestinal tract by use of the aforementioned probiotic bacterial strains with or without the concomitant administration of antibiotics. While the primary focus is on the treatment of gastrointestinal tract infections, the therapeutic compositions of the present invention may also be administered to buccal, vaginal, optic, and like physiological locations.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/148,965 filed onApr. 23, 2008 (now U.S. Pat. No. 7,807,151), which is a continuation ofU.S. Ser. No. 11/305,507, filed Dec. 16, 2005 (now U.S. Pat. No.7,708,988), which is a continuation of U.S. Ser. No. 10/264,745, filedon Oct. 4, 2002 (abandoned), which is a continuation application of U.S.Ser. No. 09/370,793, filed on Aug. 5, 1999 (now U.S. Pat. No.6,461,607), which claims priority to U.S. Ser. No. 60/097,594 filed onAug. 24, 1998, abandoned, the contents of each of these applications areherein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for theutilization of probiotic organisms in therapeutic compositions. Morespecifically, the present invention relates to the utilization of one ormore species or strains of lactic acid-producing bacteria, preferablystrains of Bacillus coagulans, for the control of gastrointestinal tractpathogens, including antibiotic-resistant gastrointestinal tractpathogens, and their associated diseases by both a reduction in the rateof colonization and the severity of the deleterious physiologicaleffects of the colonization of the antibiotic-resistant pathogen. Inaddition, the present invention relates to the utilization oftherapeutic compounds comprised of lactic acid-producing bacteria andanti-microbial agents such as antibiotics, anti-fungal compounds,anti-yeast compounds, or anti-viral compounds. In addition, the presentinvention relates to the use of lactic acid-producing bacteria inanimals to mitigate gastrointestinal tract pathogens.

BACKGROUND OF THE INVENTION

1. Probiotic Microorganisms

The gastrointestinal microflora has been shown to play a number of vitalroles in maintaining gastrointestinal tract function and overallphysiological health. For example, the growth and metabolism of the manyindividual bacterial species inhabiting the gastrointestinal tractdepend primarily upon the substrates available to them, most of whichare derived from the diet. See e.g., Gibson G. R. et al., 1995.Gastroenterology 106: 975-982; Christl, S. U. et al., 1992. Gut 33:1234-1238. These finding have led to attempts to modify the structureand metabolic activities of the community through diet, primarily withprobiotics which are live microbial food supplements. The best knownprobiotics are the lactic acid-producing bacteria (i.e., Lactobacilli)and Bifidobacteria, which are widely utilized in yogurts and other dairyproducts. These probiotic organisms are non-pathogenic andnon-toxigenic, retain viability during storage, and survive passagethrough the stomach and small intestine. Since probiotics do notpermanently colonize the host, they need to be ingested regularly forany health promoting properties to persist. Commercial probioticpreparations are generally comprised of mixtures of Lactobacilli andBifidobacteria, although yeast such as Saccharomyces have also beenutilized.

Probiotic preparations were initially systematically evaluated for theireffect on health and longevity in the early-1900's (see e.g.,Metchinikoff, E., Prolongation of Life, Willaim Heinermann, London1910), although their utilization has been markedly limited since theadvent of antibiotics in the 1950's to treat pathological microbes. Seee.g., Winberg, et al, 1993. Pediatr. Nephrol. 7: 509-514; Malin et al,Ann. Nutr. Metab. 40: 137-145; and U.S. Pat. No. 5,176,911. Similarly,lactic acid-producing bacteria (e.g., Bacillus, Lactobacillus andStreptococcus species) have been utilized as food additives and therehave been some claims that they provide nutritional and/or therapeuticvalue. See e.g., Gorbach, 1990. Ann. Med. 22: 37-41; Reid et al, 1990.Clin. Microbiol. Rev. 3: 335-344.

Therefore, probiotic microorganisms are those which confer a benefitwhen grow in a particular environment, often by inhibiting the growth ofother biological organisms in the same environment. Examples ofprobiotic organisms include bacteria and bacteriophages which possessthe ability to grow within the gastrointestinal tract, at leasttemporarily, to displace or destroy pathogenic organisms, as well asproviding other benefits to the host. See e.g., Salminen et al, 1996.Antonie Van Leeuwenhoek 70: 347-358; Elmer et al, 1996. JAMA 275:870-876; Rafter, 1995. Scand. J. Gastroenterol. 30: 497-502; Perdigon etal, 1995. J. Dairy Sci. 78: 1597-1606; Gandi, Townsend Lett. Doctors &Patients, pp. 108-110, January 1994; Lidbeck et al, 1992. Eur. J. CancerPrev. 1: 341-353.

The majority of previous studies on probiosis have been observationalrather than mechanistic in nature, and thus the processes responsiblefor many probiotic phenomena have yet to be quantitatively elucidated.Some probiotics are members of the normal colonic microflora and are notviewed as being overtly pathogenic. However, these organisms haveoccasionally caused infections (e.g., bacteremia) in individuals whoare, for example, immunocompromised. See e.g., Sussman, J. et al., 1986.Rev Infect. Dis. 8: 771-776; Hata, D. et al., 1988. Pediatr. Infect.Dis. 7: 669-671.

While the attachment of probiotics to the gastrointestinal epithelium isan important determinant of their ability to modify host immunereactivity, this is not a universal property of Lactobacilli orBifidobacteria, nor is it essential for successful probiosis. See e.g.,Fuller, R., 1989. J. Appl. Bacteriol. 66: 365-378. For example,adherence of Lactobacillus acidophilus and some Bifidobacteria to humanenterocyte-like CACO-2 cells has been demonstrated to prevent binding ofenterotoxigenic and enteropathogenic Escherichia coli, as well asSalmonella typhimurium and Yersinia pseudotuberculosis. See e.g.,Bernet, M. F. et al., 1994. Gut 35: 483-489; Bernet, M. F. et al., 1993.Appl. Environ. Microbiol. 59: 4121-4128.

While the gastrointestinal microflora presents a microbial-based barrierto invading organisms, pathogens often become established when theintegrity of the microbiota is impaired through stress, illness,antibiotic treatment, changes in diet, or physiological alterationswithin the G.I. tract. For example, Bifidobacteria are known to beinvolved in resisting the colonization of pathogens in the largeintestine. See e.g., Yamazaki, S. et al., 1982. Bifidobacteria andMicroflora 1: 55-60. Similarly, the administration of Bifidobacteriabreve to children with gastroenteritis eradicated the causativepathogenic bacteria (i.e., Campylobacter jejuni) from their stools (seee.g., Tojo, M., 1987. Acta Pediatr. Jpn. 29: 160-167) andsupplementation of infant formula milk with Bifidobacteria bifidum andStreptococcus thermophilus was found to reduce rotavirus shedding andepisodes of diarrhea in children who were hospitalized (see e.g.,Saavedra, J. M., 1994. The Lancet 344: 1046-109.

In addition, some lactic acid producing bacteria also producebacteriocins which are inhibitory metabolites which are responsible forthe bacteria's anti-microbial effects. See e.g., Klaenhammer, 1993. FEMSMicrobiol. Rev. 12: 39-85; Barefoot et al., 1993. J. Diary Sci. 76:2366-2379. For example, selected Lactobacillus strains which produceantibiotics have been demonstrated as effective for the treatment ofinfections, sinusitis, hemorrhoids, dental inflammations, and variousother inflammatory conditions. See e.g., U.S. Pat. No. 5,439,995.Additionally, Lactobacillus reuteri has been shown to produceantibiotics which possess anti-microbial activity against Gram negativeand Gram positive bacteria, yeast, and various protozoan. See e.g., U.S.Pat. Nos. 5,413,960 and 5,439,678.

Probiotics have also been shown to possess anti-mutagenic properties.For example, Gram positive and Gram negative bacteria have beendemonstrated to bind mutagenic pyrolysates which are produced duringcooking at a high temperature. Studies performed with lacticacid-producing bacteria has shown that these bacteria may be eitherliving or dead, due to the fact that the process occurs by adsorption ofmutagenic pyrolysates to the carbohydrate polymers present in thebacterial cell wall. See e.g., Zang, X. B. et al., 1990. J. Dairy Sci.73: 2702-2710. Lactobacilli have also been shown to degrade carcinogens(e.g., N-nitrosamines), which may serve an important role if the processis subsequently found to occur at the level of the mucosal surface. Seee.g., Rowland, I. R. and Grasso, P., Appl. Microbiol. 29: 7-12.Additionally, the co-administration of lactulose and Bifidobacterialongum to rats injected with the carcinogen azoxymethane wasdemonstrated to reduce intestinal aberrant crypt foci, which aregenerally considered to be pre-neoplastic markers. See e.g., Challa, A.et al., 1997. Carcinogenesis 18: 5175-21. Purified cell walls ofBifidobacteria may also possess anti-tumorigenic activities in that thecell wall of Bifidobacteria infantis induces the activation ofphagocytes to destroy growing tumor cells. See e.g., Sekine, K. et al.,1994. Bifidobacteria and Microflora 13: 65-77. Bifidobacteria probioticshave also been shown to reduce colon carcinogenesis induced by1,2-dimethylhydrazine in mice when concomitantly administered withfructo-oligosaccharides (FOS; see e.g., Koo, M. B., and Rao, A. V.,1991. Nutrit. Rev. 51: 137-146), as well as inhibiting liver and mammarytumors in rats (see e.g., Reddy, B. S., and Rivenson, A., 1993. CancerRes. 53: 3914-3918).

It has also been demonstrated that the microbiota of thegastrointestinal tract affects both mucosal and systemic immunity withinthe host. See e.g., Famularo, G. et al., Stimulation of Immunity byProbiotics. In: Probiotics: Therapeutic and Other Beneficial Effects.pg. 133-161. (Fuller, R., ed. Chapman and Hall, 1997). The intestinalepithelial cells, blood leukocytes,

B- and T-lymphocytes, and accessory cells of the immune system have allbeen implicated in the aforementioned immunity. See e.g., Schiffrin, E.J. et al., 1997. Am. J. Clin. Nutr. 66 (suppl): 5-20S. Other bacterialmetabolic products which possess immunomodulatory properties include:endotoxic lipopolysaccharide, peptidoglycans, and lipoteichoic acids.See e.g., Standiford, T. K., 1994. Infect. Linmun. 62: 119-125.Accordingly, probiotic organisms are thought to interact with the immunesystem at many levels including, but not limited to: cytokineproduction, mononuclear cell proliferation, macrophage phagocytosis andkilling, modulation of autoimmunity, immunity to bacterial and protozoanpathogens, and the like. See e.g., Matsumara, K. et al., 1992. AnimalSci. Technol. (Jpn) 63: 1157-1159; Solis-Pereyra, B. and Lemmonier, D.,1993. Nutr. Res. 13: 1127-1140. Lactobacillus strains have also beenfound to markedly effect changes in inflammatory and immunologicalresponses including, but not limited to, a reduction in colonicinflammatory infiltration without eliciting a similar reduction in thenumbers of B- and T-lymphocytes. See e.g., De Simone, C. et al., 1992.Immunopharmacol. Immunotoxicol. 14: 331-340.2. Gastrointestinal Effects of Antibiotic Administration

Antibiotics are widely used to control pathogenic microorganisms in bothhumans and animals. Unfortunately, the widespread use of anti-microbialagents, especially broad spectrum antibiotics, has resulted in a numberof serious clinical consequences. For example, antibiotics often killbeneficial, non-pathogenic microorganisms (i.e., flora) within thegastrointestinal tract which contribute to digestive function andhealth. Accordingly, relapse (the return of infections and theirassociated symptoms) and secondary opportunistic infections often resultfrom the depletion of lactic acid-producing and other beneficial florawithin the gastrointestinal tract.

Unfortunately, most, if not all, lactic acid-producing or probioticbacteria are extremely sensitive to common antibiotic compounds.Accordingly, during a normal course of antibiotic therapy, manyindividuals develop a number of deleterious physiological side-effectsincluding: diarrhea, intestinal cramping, and sometimes constipation.These side-effects are primarily due to the non-selective action ofantibiotics, as antibiotics do not possess the ability to discriminatebetween beneficial, non-pathogenic and pathogenic bacteria, bothbacterial types are killed by these agents. Thus, individuals takingantibiotics offer suffer from gastrointestinal problems as a result ofthe beneficial microorganisms (i.e., intestinal flora), which normallycolonize the gastrointestinal tract, being killed or severelyattenuated. The resulting change in the composition of the intestinalflora can result in vitamin deficiencies when the vitamin-producingintestinal bacteria are killed, diarrhea and dehydration and, moreseriously, illness should a pathogenic organism overgrow and replace theremaining beneficial gastrointestinal bacteria.

Another deleterious result of indiscriminate use of anti-microbialagents is the generation of multiple antibiotic-resistant pathogens. Seee.g., Mitchell, P. 1998. The Lancet 352: 462-463; Shannon, K., 1998.Lancet 352: 490-491. The initial reports of meticillin-resistantStaphylococcus aurous (MRSA) infections have been over-shadowed by themore recent outbreaks of vancomycin-resistant Enterococci (VRE). Thedevelopment of such resistance has led to numerous reports of systemicinfections which remained untreatable with conventional antibiotictherapies. Recently, a vancomycin—(generally regarded as an antibioticof “last resort”) resistant strain of Staphylococcus aurous wasresponsible for over 50 deaths in a single Australian hospital. Seee.g., Shannon, K., 1998. Lancet 352: 490-491.

Enterococci are currently a major nosocomial pathogen and are likely toremain as such for a long period of time. Enterococci, as well as othermicrobes, obtain antibiotic resistance genes in several different ways.For example, Enterococci emit pheromones which cause them to become“sticky” and aggregate, thus facilitating the exchange of geneticmaterial, such as plasmids (autonomously replicating, circular DNA whichoften carry the antibiotic resistance genes). In addition, someEnterococci also possess “conjugative transposons” which are DNAsequences that allow them to directly transfer resistance genes withoutplasmid intermediary. It is believed that penicillin resistance has beenconferred from Enterococci to Streptococci to Staphylococci through thislater mechanism.

Since 1989, a rapid increase in the incidence of infection andcolonization with vancomycin-resistant Enterococci (VRE) has beenreported by numerous hospitals within the United States. This increaseposes significant problems, including: (i) the lack of availableanti-microbial therapy for VRE infections, due to the fact that most VREare also resistant to the drugs which were previously used to treat suchinfections (e.g., Aminoglycosides and Ampicillin); and (ii) thepossibility that the vancomycin-resistant genes present in VRE can betransferred to other gram-positive microorganisms (e.g., Staphylococcusaureus).

An increased risk for VRE infection and colonization has also beenassociated with previous vancomycin and/or multi-anti-microbial therapy,severe underlying disease or immunosuppression, and intra-abdominalsurgery. Because Enterococci can be found within the normalgastrointestinal and female genital tracts, most enterococcal infectionshave been attributed to endogenous sources within the individualpatient. However, recent reports of outbreaks and endemic infectionscaused by Enterococci, including VRE, have indicated thatpatient-to-patient transmission of the microorganisms can occur througheither direct contact or through indirect contact via (i) the hands ofpersonnel; or (ii) contaminated patient-care equipment or environmentalsurfaces.

Accordingly, there remains a need for a highly efficacious biorationaltherapy which functions to mitigate the deleterious physiologicaleffects of digestive pathogens, including antibiotic-resistantgastrointestinal tract pathogens, in both humans and animals, by thecolonization (or re-colonization) of the gastrointestinal tract withprobiotic microorganisms, following the administration of antibiotics,anti-fungal, anti-viral, and similar agents. Additionally, a need asremains for the development of a highly efficacious biorational therapywhich functions to mitigate antibiotic-resistant digestive pathogens, inboth humans and animals, by the colonization (or re-colonization) of thegastrointestinal tract with probiotic microorganisms, following theadministration of antibiotics, anti-fungal, anti-viral, and similaragents, by functioning to reduce both the colonization rate and thepotential physiologically deleterious effects due to the colonization ofantibiotic-resistant digestive pathogens.

SUMMARY OF THE INVENTION

The present invention discloses methodologies for the selective breedingand isolation of antibiotic-resistant, lactic acid-producing bacterialstrains for utilization in various types of therapeutic applications.For example, in one specific embodiment, these lactic acid-producingbacteria are co-administered with one or more anti-microbial compounds(e.g., antibiotics, anti-mycotic compounds, anti-viral compounds, andthe like). It should be noted that, in most clinical and scientificfields, the production or evolution of antibiotic resistantmicroorganisms is an undesirable consequence of unnecessary issue and/orimproper use of antibiotics compounds. However, the present inventionserves to constructively produce bacteria that possess resistance to asingle, as opposed to multiple, antibiotics.

In another related aspect, the present invention discloses compositionsand methodologies for the utilization of these compositions comprisingnon-pathogenic, probiotic lactic acid-producing bacteria which are usedto mitigate the deleterious physiological effects of gastrointestinaltract pathogens, including antibiotic-resistant gastrointestinal tractpathogens, in both humans and animals, by the colonization (ormore-correctly, re-colonization) of the gastrointestinal tract withprobiotic microorganisms, following the administration of antibiotics,anti-fungal, anti-viral, and similar agents.

Additionally, the present invention relates to the use of lacticacid-producing bacteria to mitigate the effects of parasites andpathogens in animals.

1. Co-Administration of Probiotic Bacterial with Anti-MicrobialCompounds

It has been demonstrated that common and antibiotic resistant digestivepathogens can be controlled with the utilization of particular probioticorganisms that have been identified for their ability to remain viablein the gastrointestinal tract during antibiotic therapy. However, itshould be noted that, prior to the disclosure of the present invention,most strains of probiotic bacteria (e.g., Lactobacillus,Bifidiobacterium, and Bacillus) were found to be sensitive to themajority of antibiotics, hence they were not particularly suitable forco-administration with broad-spectrum antibiotics.

Accordingly, in the present invention, strains of Bacillus coagulanswere isolated and identified for their ability to remain viable whenexposed to typical therapeutic concentrations of antibiotics that arecommonly used to mitigate digestive pathogens. These new Bacillusvariants disclosed herein may be administered prior to, concomitantlywith, or subsequent to the administration of antibiotics. In a preferredembodiment, these Bacillus strains are co-administered in combinationwith the selected antibiotic which they are resistant to.

One probiotic bacterial strain disclosed by the present invention isBacillus coagulans GB-M—a new variant or mutant of Bacillus coagulansATCC No. 31284. Bacillus coagulans GB-M has been demonstrated to beresistant to Macrolide antibiotics such as Azithromycin, Erythromycinand other similar antibiotic compounds. The advantages of using abiological in combination with a chemical antibiotic or the concurrentuse of a biological with a chemical serves to address the many hazardsand side effects of antibiotic therapy. In addition, the use of theseaforementioned variants, as well as other lactic acid-producingbiorationals, in combination with chemotherapy drugs and anti-fungalwould be of great benefit to those taking these compounds, due to thefact that these individuals, more often than not, suffer from sideeffects which are a direct result of depleted “normal” gastrointestinalflora.

In addition to the aforementioned aspects of the present invention, theutilization of bifidogenic oligosaccharides (e.g.,fructo-oligosaccharides (FOS)) are beneficial to facilitate there-establishment and proliferation of other beneficial lacticacid-producing bacteria and to further promote gastrointestinalmicrobial biodiversity. In one embodiment of the present invention, acomposition comprising an isolated and specific antibiotic resistantBacillus coagulans strain in combination with an effective amount of afructo-oligosaccharide (FOS) in a pharmaceutically acceptable carriersuitable for administration to the gastrointestinal track of a human oranimal is disclosed. In preferred embodiments of the present invention,the Bacillus coagulans strain is included in the composition in the formof spores, a dried cell mass, in the form of a flowable concentrate, orin the form of a stabilized gel or paste.

In another embodiment of the present invention, the Bacillus coagulansstrain is combined with a therapeutically-effective dose of anantibiotic. In preferred embodiments of the present invention, theBacillus coagulans strain is combined with a therapeutic concentrationof antibiotic including, but not limited to: Gentamicin; Vancomycin;Oxacillin; Tetracyclines; Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-sulfamethoxasole; a member of the Cephlosporin antibioticfamily (e.g., Cefaclor, Cefadroxil, Cefixime, Cefprozil, Ceftriaxone,Cefuroxime, Cephalexin, Loracarbef, and the like); a member of thePenicillin family of antibiotics (e.g., Ampicillin,Amoxicillin/Clavulanate, Bacampicillin, Cloxicillin, Penicillin VK, andthe like); with a member of the Fluoroquinolone family of antibiotics(e.g., Ciprofloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like); or amember of the Macrolide antibiotic family (e.g., Azithromycin,Erythromycin, and the like).

Similarly, a therapeutically-effective concentration of an anti-fungalagent may also be utilized. Such anti-fungal agents include, but are notlimited to: Clotrimazole, Fluconazole, Itraconazole, Ketoconazole,Miconazole, Nystatin, Terbinafine, Terconazole, and Tioconazole.

The aforementioned embodiment involves selectively-culturing theprobiotic bacteria (which may initially be sensitive to the antibioticof choice) in gradually increasing concentrations of antibiotic in orderto facilitate the development of decreased antibiotic sensitivity or,preferably, total antibiotic resistance. It should be noted that this isthe most preferred embodiment of the present invention due to the factthat current FDA (and other governmental agency) regulations expresslyprohibit the intentional release of recombinant antibiotic resistantbacterial strains into the environment. Hence, the utilization of theantibiotic resistant strains of bacteria disclosed in the presentinvention, produced through non-recombinant methodologies, would not beviolative of these aforementioned regulations.

Similarly, further embodiments of the present invention disclosesmethodologies for the generation of antibiotic-resistant strains oflactic acid-producing bacteria by microbial genetic- and recombinantDNA-based techniques. With respect to the microbial genetic-basedmethodology antibiotic resistance may be conferred by the “transfer” ofgenetic information from an antibiotic resistant bacterial strain to anantibiotic sensitive bacterial strain through plasmid- andnon-plasmid-mediated genetic transfer. Plasmids are small,non-chromosomal, autonomously replicating, circular DNA which oftencarry the antibiotic resistance genes. For example, in one embodiment ofthe present invention, conjugative transposons (i.e., DNA sequences thatallow the direct transfer of resistance genes without a plasmidintermediary) may be utilized to confer antibiotic resistance to anantibiotic sensitive bacterial stain. In another embodiment, recombinantDNA-based, plasmid-mediated methodologies may also be utilized.

These novel, antibiotic resistant bacterial isolates will then be usedin combination with an appropriate antibiotic for the mitigation ofpathogen-associated disease and/or the re-establishment of normaldigestive flora following the administration of antibiotics and/or otheragents which deplete the gastrointestinal ecology. Hence, the presentinvention demonstrates that all antibiotic compounds possess the abilityto work synergistically with an antibiotic-resistant biorational toincrease the overall efficacy of antibiotic administration, whileconcomitantly mitigating deleterious side-effects.

In another embodiment of the present invention, the beneficial,antibiotic resistant, lactic acid-producing bacterial strain isco-administered with an anti-fungal agent and/or an antibiotic so as toameliorate the growth of both the mycotic and/or bacterial pathogen. Inaddition, anti-viral agents, as well as agents which inhibit the growthof yeast may also be utilized, with or without the concomitantadministration of an antibiotic.

In yet another embodiment of the present invention, the administrationof the beneficial, lactic acid-producing bacterial strain is, by way ofexample but not of limitation, topical, vaginal, intra-ocular,intra-nasal, intra-otic, buccal, and the like.

2. Use of Probiotic Bacteria to Inhibit Colonization ofAntibiotic-Resistant Gastrointestinal Pathogens

Additionally disclosed herein are compositions and methods of treatmentwhich exploit the novel discovery that specific, lactic acid-producingbacteria (e.g., Bacillus coagulans) possess the ability to exhibitinhibitory activity in preventing and reducing the colonization rates ofgastrointestinal bacterial infections, particularly those infectionsassociated with antibiotic resistant pathogens such as Enterococccus,Clostridium, Escherichia, and Staphylococcus species, as well asmitigating the deleterious physiological effects of the infection by thepathogen. Exceptionally hardy or enteric-coated lactic acid-producingbacterium are preferably used, with spore-forming Bacillus species,particularly Bacillus coagulans, being a preferred embodiment. Thepresent invention also discloses therapeutic compositions, therapeuticsystems, and methods of use for the treatment and/or prevention ofvarious pathogenic bacterial gastrointestinal tract infections,particularly those infections associated with antibiotic-resistantpathogens.

In one embodiment of the present invention, a therapeutic compositioncomprising a viable, non-pathogenic lactic acid-producing bacterium,preferably Bacillus coagulans, in a pharmaceutically-acceptable carriersuitable for oral administration to the gastrointestinal tract of ahuman or animal, is disclosed. In another embodiment, a Bacilluscoagulans strain is included in the therapeutic composition in the formof spores. In another embodiment, a Bacillus coagulans strain isincluded in the composition in the form of a dried cell mass.

In another aspect of the present invention, a composition s comprisingan extracellular product of a lactic acid-producing bacterial strain,preferably Bacillus coagulans, in a pharmaceutically-acceptable carriersuitable for oral administration to a human or animal, is disclosed. Ina preferred embodiment, the extracellular product is a supernatant orfiltrate of a culture of an isolated Bacillus coagulans strain.

Another aspect of the invention is a method of preventing or treating abacterial gastrointestinal infection in a human, comprising the steps oforally administering to a human subject a food or drink formulationcontaining viable colony forming units of a non-pathogenic lactic acidbacterium, preferably a Bacillus species and more preferably an isolatedBacillus coagulans strain, and allowing the bacteria to grow in thehuman subject's gastrointestinal tract.

In one embodiment of the aforementioned method, the step of allowing thenon-pathogenic bacteria to grow, further includes inhibiting growth ofantibiotic-resistant Candida species, Staphylococcus species,Streptococcus species, Proteus species, Pseudomonas species, Escherichiacoli, Clostridium species, Klebsiella species, and Enterococccusspecies. In a preferred embodiment, the method inhibitsantibiotic-resistant Pseudomonas aeruginosa, Staphylococcus aureus,Staphylococcus pyogenes, Clostridium perfingens, Clostridium dificile,Clostridium botulinum, Clostridium tributrycum, Clostridium sporogenes,Enterococccus faecalis, Enterococccus faecium, and various othersignificant species of antibiotic gastrointestinal pathogens orcombinations thereof.

One aspect of the invention is a lactic acid-producing bacterialcomposition comprising an isolated Bacillus species strain, combinedwith a pharmaceutically-acceptable carrier suitable for oraladministration to a human or animal, wherein the isolated Bacillusspecies strain is capable of growing at temperatures of about 30° C. toabout 65° C., produces L(+) dextrorotatory lactic acid, produces sporesresistant to heat up to 90° C., and exhibits competitive, antibiotic, orparasitical activity that inhibits or reduces the colonization rate ofthe pathogenic bacteria associated with gastroenteritis and othersignificant digestive pathogens. The probiotic activity primarilyresults from vegetative growth of the isolated Bacillus species strainin the gastrointestinal tract of a human or animal. This growth causes adirect competition with the pathogenic bacteria, as well as producing anacidic, non-hospitable environment. In yet another embodiment, theprobiotic activity results from an extracellular product of the isolatedlactic acid-producing strain produced within the gastrointestinal. Thepresent invention also discloses a therapeutic system for treating,reducing or controlling gastrointestinal bacterial infections,particularly infections associated with antibiotic-resistant pathogens.

The present invention provides several advantages. In particular,insofar as there is a detrimental effect to the use of antibioticsbecause of the potential to produce antibiotic-resistant microbialspecies, it is desirable to have an anti-microbial therapy which doesnot utilize conventional anti-microbial agents. Hence, the presentinvention does not contribute to the production of future generations ofantibiotic-resistant pathogens.

3. Use of Probiotic Bacteria in Animals

It has now been discovered that parasites and pathogens colonizing theintestinal tract of animals can be inhibited and/or controlled by theuse of diatomaceous earth in combination with the use of a probioticlactic acid producing bacteria.

The present invention describes compositions, therapeutic systems, andmethods of use for inhibiting pathogen and/or parasite growth in thegastrointestinal tract and feces of animals. A composition of thisinvention comprises an effective amount of diatomaceous earth incombination with a non-pathogenic lactic acid-producing bacteria, withspore-forming Bacillus species, particularly Bacillus coagulans, being apreferred embodiment.

According to the invention, there is provided a composition comprisingdiatomaceous earth in combination with a lactic acid-producing bacteriain a pharmaceutically- or nutritionally-acceptable carrier suitable fororal administration to the digestive tract of an animal. In oneembodiment of the composition, a Bacillus coagulans strain is includedin the composition in the form of spores. In another embodiment, aBacillus coagulans strain is included in the composition in the form ofa dried cell mass. In another embodiment, a Bacillus coagulans strain isincluded in the composition in the form of a stabilized paste. Inanother embodiment, a Bacillus coagulans strain is included in thecomposition in the form of stabilized gel. In another embodiment, aBacillus coagulans strain is included in the composition in the form ofa stabilized liquid suspension.

In one embodiment, the invention contemplates a composition comprisingdiatomaceous earth comprised predominantly of the Melosira genus,preferably at least 80%. In one embodiment, the bacterial is present inthe composition at a concentration of approximately 1×10³ to 1×10¹⁴colony forming units (CFU)/gram, preferably approximately 1×10⁵ to1×10¹² CFU/gram, whereas in other preferred embodiments theconcentrations are approximately 1×10⁹ to 1×10¹³ CFU/gram, approximately1×10⁵ to 1×10⁷ CFU/g, or approximately 1×10⁸ to 1×10⁹ CFU/gram.

In one embodiment, the bacteria is in a pharmaceutically acceptablecarrier suitable for oral administration to an animal, preferably, as apowdered food supplement, a variety of pelletized formulations, or aliquid formulation. In one embodiment, the composition further includesan effective amount of a bifidogenic oligosaccharide, such as a short orlong chain fructo-oligosaccharide (FOS), a gluco-oligosaccharide (GOS)or other long-chain oligosaccharide polymer not readily digested bypathogenic bacteria as described herein.

The invention also describes a therapeutic system for inhibitingpathogen and/or parasite growth in the gastrointestinal tract and/orfeces of an animal comprising a container comprising a label and acomposition as described herein, wherein said label comprisesinstructions for use of the composition for inhibiting pathogen and/orparasite growth.

It should be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the present invention as claimed.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates, in tabular form, a summary of the metabolic growthcharacteristics and requirements of Bacillus coagulans.

FIG. 2 illustrates, in tabular form, the ability of Bacillus coagulansto inhibit various fungal pathogens, of the Trichophyton species, usingan in vitro assay. The ATCC Accession Numbers of each fungal strain ofthe Trichophyton species from the American Type Culture Collection(ATCC) are also enumerated herein.

FIG. 3 illustrates, in tabular form, the ability of Bacillus coagulansto inhibit various yeast pathogens, of the Candida species, using an invitro assay. The ATCC Accession Numbers of each yeast strain of theCandida species from the American Type Culture Collection (ATCC) arealso enumerated herein.

FIG. 4 illustrates, in tabular form, Formulations 1-6 of the therapeuticcompositions of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in therelevant art. Unless mentioned otherwise, the techniques employed orcontemplated herein are standard methodologies well known to one ofordinary skill in the art. The examples of embodiments are forillustration only.

The present invention is discloses the recent discovery thatnon-pathogenic, lactic acid-producing bacterial species (i.e.,“probiotic bacteria”), such as the exemplary Bacillus coagulans, may beutilized in combination with antibiotic compounds or other functionalanti-microbial drugs and supplements so as to form therapeuticcompositions for use in ameliorating and/or controlling the colonizationof pathogenic bacteria with the gastrointestinal tract of both humansand animals. In addition, these non-pathogenic, lactic-producing,probiotic bacteria may be co-administered with an anti-fungal agentand/or an antibiotic to ameliorate the growth of the mycotic orbacterial pathogen in question. In brief, the present invention utilizesantibiotic-resistant, non-pathogenic bacteria to mitigate the growth andsubsequent establishment of antibiotic-resistant, pathogenic microbeswithin, for example, the gastrointestinal tract. Also disclosed hereinare various therapeutic compositions, methods for using said therapeuticcompositions, and systems for containing and administering/deliveringsaid therapeutic compositions.

In addition, the present invention the present invention also disclosescompositions and methodologies for the utilization of thesecompositions, comprising non-pathogenic, probiotic lactic acid-producingbacteria, in the mitigation of the deleterious physiological effects ofgastrointestinal tract pathogens, including antibiotic-resistantgastrointestinal tract pathogens, in both humans and animals, by thecolonization (or more-correctly, re-colonization) of thegastrointestinal tract with probiotic microorganisms, following theadministration of antibiotics, anti-fungal, anti-viral, and similaragents.

1. Antibiotic Administration and Biorational Therapy

Antibiotics are widely used to control pathogenic microorganisms in bothhumans and animals. Unfortunately, the indiscriminate use of theseagents has led to the generation of pathogenic bacteria which frequentlyexhibit resistance to multiple antibiotics. In addition, theadministration of antibiotics often results in the killing of many ofthe beneficial microorganisms (i.e., flora) within the gastrointestinaltract which contribute to “normal” gastrointestinal function (e.g.,digestion, absorption, vitamin production, and the like). Accordingly,relapse (the return of infections and their associated symptoms) andsecondary opportunistic infections often result from the depletion ofLactobacillus and other types of beneficial microbial flora within thegastrointestinal tract. Unfortunately, most, if not all, lacticacid-producing or probiotic bacteria are extremely sensitive to commonantibiotic compounds. Therefore, during a normal course of antibiotictherapy, many individuals develop a number of deleterious physiologicalside-effects including: diarrhea, intestinal cramping, and sometimesconstipation. These side-effects are primarily due to the non-selectiveaction of antibiotics, as antibiotics do not possess the ability todiscriminate between beneficial bacteria and pathogenic bacteria. Hence,both pathogenic and non-pathogenic bacteria are killed by these agents.

A biorational therapy that includes an antibiotic and an appropriatemicroorganism that is resistant to the selected antibiotic would serveto enhance the efficacy of the antibiotic (if the antibiotic is used forthe purpose of controlling a gastrointestinal tract pathogen) and assistin providing a digestive environment which is conducive to thereestablishment of the endogenous lactic acid bacteria and suppress thegrowth of pathogens.

It should also be noted that the present invention is not limited solelyto oral administration of the therapeutic compounds disclosed herein.For example, antibiotic and anti-fungal resistance is also associatedwith topical and intra-vaginal medications. Thus, in an additionalembodiment, the co-administration of a lactic acid or other beneficialbacterial culture with a vaginal anti-fungal medication wouldeffectively aid in the mitigation of the mycotic or bacterial pathogenin question and repopulate the vagina and reduce the incidence ofrelapse. It should be noted that it has been demonstrated that theabsence of lactic acid-producing bacteria within the vagina is the mostcommon etiology of vaginal yeast infections and bacterial vaginosis.

In an additional embodiment, skin creams, lotions, gels and the likecould similarly contain a beneficial biorational component that would beeffective in controlling pathogenic organisms on the skin and furtherreduce the emergence of antibiotic resistant pathogens. By way ofexample, but not of limitation, the cells, spores or extracellularmaterials from such beneficial biorational bacteria could beincorporated into these skin products for this express purpose. Burnpatients usually are given antibiotics to reduce the incidence ofopportunistic infection. Pathogenic Pseudomonas, Staphylococcus, and/orEnterococci are frequently associated with infections of severe burns.Hence, the salves, lotions, gels and the like combined with thebeneficial, biorational microorganisms or their extracellular products,as disclosed in the present invention, would be effective in achieving astate of proper biodiversity to the skin in burn cases, as, generally,such biodiversity is not associated with pathogenic overgrowth.

A further embodiment of the present invention involves the utilizationof probiotic organisms in livestock production, in which antibioticssuch as Vancomycin and Gentamicin are commonly used to stimulate healthand weight gain. Most, if not all, probiotic organisms are sensitive tothese two antibiotics and this fact has limited the potential use ofsuch microorganisms in the livestock industry. In addition, there aremany environmentally-related problems associated with the use ofantibiotics in livestock production. For example, antibiotic ladenanimal waste degrades very slowly and the antibiotic residue canpersist, further slowing biodegradation. With the addition of species ofbacteria that are resistant to Vancomycin, Gentamicin, and otherantibiotics, biodegradation could actually be enhanced.

2. Probiotic, Lactic Acid-Producing Bacterial Strains

A biorational therapy which includes an antibiotic and an appropriatemicroorganism that is resistant to the selected antibiotic serves toboth enhance the overall therapeutic efficacy of the antibiotic (if theantibiotic is used for the purpose of controlling a digestive pathogen)and to assist in providing a gastrointestinal environment that isconducive to the reestablishment of the endogenous lactic acid-producingbacteria and to suppress the concomitant growth of pathogenicmicroorganisms.

As utilized herein, “probiotic” refers to microorganisms that form atleast a part of the transient or endogenous flora and thereby exhibit abeneficial prophylactic and/or therapeutic effect on the host organism.Probiotics are generally known to be clinically safe (i.e.,non-pathogenic) by those individuals skilled in the art. By way ofexample, and not of limitation to any particular mechanism, theprophylactic and/or therapeutic effect of a lactic acid-producingbacteria of the present invention results, in part, from a competitiveinhibition of the growth of pathogens due to: (i) their superiorcolonization abilities; (ii) parasitism of undesirable microorganisms;(iii) the production of lactic acid and/or other extracellular productspossessing anti-microbial activity; or (iv) various combinationsthereof. It should be noted that the aforementioned products andactivities of the lactic acid-producing bacteria of the presentinvention act synergistically to produce the beneficial probiotic effectdisclosed herein.

A probiotic bacteria which is suitable for use in the methods andcompositions of the present invention: (i) possesses the ability toproduce lactic acid; (ii) demonstrates beneficial function within thegastrointestinal tract; and is non-pathogenic. By way of example and notof limitation, many suitable bacteria have been identified and aredescribed herein, although it should be noted that the present inventionis not to be limited to currently-classified bacterial species insofaras the purposes and objectives as disclosed. The physiochemical resultsfrom the in vivo production of lactic acid is key to the effectivenessof the probiotic lactic acid-producing bacteria of the presentinvention. Lactic acid production markedly decreases the pH (i.e.,increases acidity) within the local micro-floral environment and doesnot contribute to the growth of many undesirable,physiologically-deleterious bacteria and fungi. Thus, by the mechanismof lactic acid production, the probiotic inhibits growth of competingpathogenic bacteria.

Typical lactic acid-producing bacteria useful as a probiotic of thisinvention are efficient lactic acid producers which includenon-pathogenic members of the Bacillus genus which produce bacteriocinsor other compounds which inhibit the growth of pathogenic organisms.Exemplary lactic acid-producing, non-pathogenic Bacillus speciesinclude, but are not limited to: Bacillus coagulans; Bacillus coagulansHammer; and Bacillus brevis subspecies coagulans.

Exemplary lactic acid-producing Lactobacillus species include, but arenot limited to: Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus DDS-1, Lactobacillus GG, Lactobacillus rhamnosus,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus gasserii,Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus,bulgaricus, Lactobacillus salivarius and Lactobacillus sporogenes (alsodesignated as Bacillus coagulans).

Exemplary lactic acid-producing Sporolactobacillus species include allSporolactobacillus species, for example, Sporolactobacillus P44.

Exemplary lactic acid-producing Bifidiobacterium species include, butare not limited to: Bifidiobacterium adolescentis, Bifidiobacteriumanimalis, Bifidiobacterium bifidum, Bifidiobacterium bifidus,Bifidiobacterium breve, Bifidiobacterium infantis, Bifidiobacteriuminfantus, Bifidiobacterium longum, and any genetic variants thereof.

Several Bacillus species which are preferred in the practice of thepresent invention, include, but are not limited to the lacticacid-producing Bacillus coagulans and Bacillus laevolacticus. Variousother non-lactic acid-producing Bacillus species may be utilized in thepresent invention so long as they produce compounds which possess theability to inhibit pathogenic bacterial or mycotic growth. Examples ofsuch suitable non-lactic acid-producing Bacillus include, but are notlimited to: Bacillus subtilis, Bacillus unifiagellatus, Bacilluslateropsorus, Bacillus laterosporus BOD, Bacillus megaterium, Bacilluspolymyxa, Bacillus licheniformis, Bacillus pumilus, and Bacillussterothermophilus. Other strains that could be employed due to probioticactivity include members of the Streptococcus (Enterococcus) genus. Forexample, Enterococcus faecium, is commonly used as a livestock probioticand, thus, could be utilized as a co-administration agent. It should benoted that, although exemplary of the present invention, Bacilluscoagulans is only utilized herein as a model for various otheracid-producing (e.g., lactic acid) species of probiotic bacteria whichmay be useful in the practice of the present invention, and therefore isnot to be considered as limiting. Furthermore, it is also intended thatany of the acid-producing species of probiotic or nutritional bacteriacan be used in the compositions, therapeutic systems and methods of thepresent invention.

The Bacillus species, particularly those species having the ability toform spores (e.g., Bacillus coagulans), are a preferred embodiment ofthe present invention. The ability to sporulate makes these bacterialspecies relatively resistant to heat and other conditions, provides fora long shelf-life in product formulations, and is deal for survival andcolonization of tissues under conditions of pH, salinity, and the likewithin the gastrointestinal tract. Moreover, additional usefulproperties of many Bacillus species include being non-pathogenic,aerobic, facultative and heterotrophic, thus rendering these bacterialspecies safe and able to readily colonize the gastrointestinal tract.

Exemplar methods and compositions are described herein using Bacilluscoagulans ATCC No. 31284 (and new variants or mutants thereof) as aprobiotic. Purified Bacillus coagulans is particularly useful as aprobiotic in the present invention as it is generally accepted that thevarious “classic” Lactobacillus and/or Bifidiobacterium species areunsuitable for colonization of the gut due to their instability in thehighly acidic environment of the gastrointestinal tract, particularlythe human gastrointestinal tract. In contrast, the preferred Bacillusspecies of the present invention are able to survive and colonize thegastrointestinal tract in a highly efficacious manner. Additionally,probiotic Bacillus coagulans is non-pathogenic and is generally regardedas safe (i.e., GRAS classification) by the U.S. Federal DrugAdministration (FDA) and the U.S. Department of Agriculture (USDA), andby those individuals skilled within the art.

Because Bacillus coagulans possesses the ability to produceheat-resistant spores, it is particularly useful for makingpharmaceutical compositions which require heat and pressure in theirmanufacture. Accordingly, formulations that include the utilizationviable Bacillus coagulans spores in a pharmaceutically-acceptablecarrier are particularly preferred for making and using compositionsdisclosed in the present invention.

The growth of these various Bacillus species to form cell cultures, cellpastes, and spore preparations is generally well-known within the art.It should be noted that the exemplary culture and preparative methodswhich are described herein for Bacillus coagulans may be readilyutilized and/or modified for growth and preparation of the other(lactic) acid-producing bacteria disclosed in the present invention.

3. Characteristics and Sources of Bacillus coagulans

The Gram positive rods of Bacillus coagulans have a cell diameter ofgreater than 1.0 μm with variable swelling of the sporangium, withoutparasporal crystal production. Bacillus coagulans is a non-pathogenic,Gram positive, spore-forming bacteria that produces L(+) lactic acid(dextrorotatory) under homo-fermentation conditions. It has beenisolated from natural sources, such as heat-treated soil samplesinoculated into nutrient medium (see e.g., Bergey's Manual of SystemicBacteriology, Vol. 2, Sneath, P. H. A. et al., eds., Williams & Wilkins,Baltimore, Md., 1986). Purified Bacillus coagulans strains have servedas a source of enzymes including endonucleases (e.g., U.S. Pat. No.5,200,336); amylase (U.S. Pat. No. 4,980,180); lactase (U.S. Pat. No.4,323,651) and cyclo-malto-dextrin glucano-transferase (U.S. Pat. No.5,102,800). Bacillus coagulans has also been utilized to produce lacticacid (U.S. Pat. No. 5,079,164). A strain of Bacillus coagulans (alsoreferred to as Lactobacillus sporogenes; Sakaguti & Nakayama, ATCC No.31284) has been combined with other lactic acid producing bacteria andBacillus natto to produce a fermented food product from steamed soybeans(U.S. Pat. No. 4,110,477). Bacillus coagulans strains have also beenused as animal feeds additives for poultry and livestock to reducedisease and improve feed utilization and, therefore, to increase growthrate in the animals (International PCT Pat. Applications No. WO 9314187and No. WO 9411492). In particular, Bacillus coagulans strains have beenused as general nutritional supplements and agents to controlconstipation and diarrhea in humans and animals.

The purified Bacillus coagulans bacteria utilized in the presentinvention are available from the American Type Culture Collection (ATCC,Rockville, Md.) using the following accession numbers: Bacilluscoagulans Hammer NRS 727 (ATCC No. 11014); Bacillus coagulans Hammerstrain C (ATCC No. 11369); Bacillus coagulans Hammer (ATCC No. 31284);and Bacillus coagulans Hammer NCA 4259 (ATCC No. 15949). PurifiedBacillus coagulans bacteria are also available from the DeutscheSarumlung von Mikroorganismen and Zellkuturen GmbH (Braunschweig,Germany) using the following accession numbers: Bacillus coagulansHammer 1915 (DSM No. 2356); Bacillus coagulans Hammer 1915 (DSM No.2383, corresponds to ATCC No. 11014); Bacillus coagulans Hammer (DSM No.2384, corresponds to ATCC No. 11369); and Bacillus coagulans Hammer (DSMNo. 2385, corresponds to ATCC No. 15949). Bacillus coagulans bacteriacan also be obtained from commercial suppliers such as SabinsaCorporation (Piscataway, N.J.) or K.K. Fermentation (Kyoto, Japan).

These aforementioned Bacillus coagulans strains and their growthrequirements have been described previously (see e.g., Baker, D. et al,1960. Can. J. Microbiol. 6: 557-563; Nakamura, H. et al, 1988. Int. J.Svst. Bacteriol. 38: 63-73. In addition, various strains of Bacilluscoagulans can also be isolated from natural sources (e.g., heat-treatedsoil samples) using well-known procedures (see e.g., Bergey's Manual ofSystemic Bacteriology, Vol. 2, p. 1117, Sneath, P. H. A. et al., eds.,Williams & Wilkins, Baltimore, Md., 1986).

It should be noted that Bacillus coagulans had previously beenmis-characterized as a Lactobacillus in view of the fact that, asoriginally described, this bacterium was labeled as Lactobacillussporogenes (See Nakamura et al. 1988. Int. J. Syst. Bacteriol. 38:63-73). However, initial classification was incorrect due to the factthat Bacillus coagulans produces spores and through metabolism excretesL(+)-lactic acid, both aspects which provide key features to itsutility. Instead, these developmental and metabolic aspects requiredthat the bacterium be classified as a lactic acid bacillus, andtherefore it was re-designated. In addition, it is not generallyappreciated that classic Lactobacillus species are unsuitable forcolonization of the gut due to their instability in the harsh (i.e.,acidic) pH environment of the bile, particularly human bile. Incontrast, Bacillus coagulans is able to survive and colonize thegastrointestinal tract in the bile environment and even grown in thislow pH range. In particular, the human bile environment is differentfrom the bile environment of animal models, and heretofore there has notbeen any accurate descriptions of Bacillus coagulans growth in humangastrointestinal tract models.

3.1 Culture of Vegetative Bacillus coagulans

Bacillus coagulans is aerobic and facultative, and is typically culturedat pH 5.7 to 6.8, in a nutrient broth containing up to 2% (by wt) NaCl,although neither NaCl, nor KCl are required for growth. A pH of about4.0 to about 7.5 is optimum for initiation of sporulation (i.e., theformation of spores). The bacteria are optimally grown at 30° C. to 45°C., and the spores can withstand pasteurization. Additionally, thebacteria exhibit facultative and heterotrophic growth by utilizing anitrate or sulfate source. The metabolic characteristics of Bacilluscoagulans are summarized in FIG. 1.

Bacillus coagulans can be cultured in a variety of media, although ithas been demonstrated that certain growth conditions are moreefficacious at producing a culture which yields a high level ofsporulation. For example, sporulation is demonstrated to be enhanced ifthe culture medium includes 10 mg/l of MgSO₄ sulfate, yielding a ratioof spores to vegetative cells of approximately 80:20. In addition,certain culture conditions produce a bacterial spore which contains aspectrum of metabolic enzymes particularly suited for the presentinvention (i.e., production of lactic acid and enzymes for the enhancedprobiotic activity and biodegradation). Although the spores produced bythese aforementioned culture conditions are preferred, various othercompatible culture conditions which produce viable Bacillus coagulansspores may be utilized in the practice of the present invention.

Suitable media for the culture of Bacillus coagulans include: PDB(potato dextrose broth); TSB (tryptic soy broth); and NB (nutrientbroth), which are all well-known within the field and available from avariety of sources. In one embodiment of the present invention, mediasupplements which contain enzymatic digests of poultry and/or fishtissue, and containing food yeast are particularly preferred. Apreferred supplement produces a media containing at least 60% protein,and about 20% complex carbohydrates and 6% lipids. Media can be obtainedfrom a variety of commercial sources, notably DIFCO (Newark, N.J.); BBL(Cockeyesville, Md.); Advanced Microbial Systems (Shakopee, Minn.); andTroy Biologicals (Troy, Md.

In a preferred embodiment of the present invention, a culture ofBacillus coagulans Hammer bacteria (ATCC No. 31284) was inoculated andgrown to a cell density of about 1×10⁸-10⁹ cells/ml in nutrient brothcontaining: 5.0 g Peptone; 3.0 g Meat Extract; 10-30 mg MnSO₄ and 1,000ml distilled water, the broth was then adjusted to pH 7.0. The bacteriawere cultured by utilization of a standard airlift fermentation vesselat 30° C. The range of MnSO₄ acceptable for sporulation was found to be1.0 mg/l to 1.0 g/l. The vegetative bacterial cells can activelyreproduce up to 65° C., and the spores are stable up to 90° C.

Following culture, the Bacillus coagulans Hammer bacterial cells orspores were collected using standard methods (e.g., filtration,centrifugation) and the collected cells and spores may subsequently belyophilized, spray dried, air dried or frozen. As described herein, thesupernatant from the cell culture can be collected and used as anextracellular agent secreted by Bacillus coagulans which possessesanti-microbial activity useful in a formulation of this invention.

A typical yield obtained from the aforementioned culture methodology isin the range of approximately 1×10⁹ to 1×10¹³ viable cells/spores and,more typically, approximately 1×10¹¹ to 1.5×10¹¹ cells/spores per gramprior to being dried. It should also be noted that the Bacilluscoagulans spores, following a drying step, maintain at least 90%viability for up to 7 years when stored at room temperature. Hence, theeffective shelf-life of a composition containing Bacillus coagulansHammer spores at room temperature is approximately 10 years.

3.2 Preparation of Bacillus coagulans Spores

Alternately, a culture of dried Bacillus coagulans Hammer bacteria (ATCCNo. 31284) spores was prepared as follows. Approximately 1×10⁷ sporeswere inoculated into one liter of culture medium containing: 24 g(wt./vol.) potato dextrose broth; 10 g of an enzymatic-digest of poultryand fish tissue; 5 g of fructo-oligosaccharides (FOS); and 10 g MnSO₄.The culture was maintained for 72 hours under a high oxygen environmentat 37° C. so as to produce a culture having approximately 15×10¹⁰cells/gram of culture. The culture was then filtered to remove theliquid culture medium and the resulting bacterial pellet was resuspendedin water and lyophilized. The lyophilized bacteria were ground to a fine“powder” by use of standard good manufacturing practice (GMP)methodologies.

It should be noted that the most preferred embodiment of the presentinvention utilizes Bacillus coagulans in spore, rather than vegetativebacterial form.

3.3 Preparation of B. coagulans Extracellular Products

Although the primary focus of the present invention is upon theutilization of lactic acid-producing probiotic bacteria in the form ofvegetative cells or spores, an additional embodiment utilizesextracellular products comprising a supernatant or filtrate of a cultureof a Bacillus coagulans strain for the prevention and/or control ofinfections caused by bacterium, fungi, yeast, and virus, andcombinations thereof. Extracellular products of Bacillus coagulans mayalso be included in compositions such as foods and liquids to be fed toinfants.

One liter cultures of Bacillus coagulans was prepared as described inSection 5.1, except that the fructo-oligosaccharide (FOS) was omitted.The culture was maintained for 5 days as described, at which time FOSwas added at a concentration of 5 g/liter, and the culture wascontinued. Subsequently, 20 ml of carrot pulp was then added at day 7,and the culture was harvested when the culture became saturated (i.e.,no substantial cell division).

The culture was first autoclaved for 30 minutes at 250° F., and thencentrifuged at 4000 r.p.m. for 15 mm. The resulting supernatant wascollected and filtered in a Buchner funnel through a 0.8 μm filter. Thefiltrate was collected and further filtered through a 0.2 μm Nalgevacuum filter. The resulting final filtrate was then collected (anapproximate volume of 900 ml) to form a liquid containing anextracellular product which may be further purified and/orquantitatively analyzed by use of various methodologies which arewell-known within the art.

4 Bifidogenic Oligosaccharides

Bifidogenic oligosaccharides, as designated herein, are a class ofcarbohydrates particularly useful for preferentially promoting thegrowth of a lactic acid-producing bacteria of the present invention.These oligosaccharides include, but are not limited to:fructo-oligosaccharides (FOS); gluco-oligosaccharides (GOS); otherlong-chain oligosaccharide polymers of fructose and/or glucose; and thetrisaccharide-raffinose. All of these aforementioned carbohydrates arenot readily digested by pathogenic bacteria. Thus. the preferentialgrowth of lactic acid-producing bacteria is promoted by the utilizationof these bifidogenic oligosaccharides due to the nutrient requirementsof this class of bacterium, as compared to pathogenic bacteria.

Bifidogenic oligosaccharides are long chain polymers that are utilizedalmost exclusively by the indigenous Bifidobacteria and Lactobacillus inthe intestinal tract and can be similarly utilized by Bacillus. Incontrast, physiologically deleterious bacteria such as Clostridium,Staphylococcus, Salmonella and Escherichia coli cannot metabolize FOS,or other bifidogenic oligosaccharides, and therefor use of thesebifidogenic oligosaccharides in combination with a lactic acid-producingbacteria of the present, preferably Bacillus coagulans, allows thesebeneficial, probiotic bacteria to grow and effectively compete with, andeventually replace any undesirable, pathogenic microorganisms within thegastrointestinal tract.

The use of bifidogenic oligosaccharides in the compositions of thepresent invention provides a synergistic effect thereby increasing theeffectiveness of the probiotic-containing compositions disclosed herein.This synergy is manifested by selectively increasing the ability of theprobiotic bacterium to grow by, for example, increasing the level ofnutrient supplementation which preferentially selects for growth of theprobiotic bacteria over many other bacterial species within the infectedtissue.

In addition, it is readily understood that Bifidobacteria andLactobacillus are also producers of lactic acid. Bifidogenicoligosaccharides enable these aforementioned probiotic organisms toproliferate preferentially over the undesirable bacteria within thegastrointestinal tract, thereby augmenting the probiotic state of thebody by further enhancing the solubility of these nutrients (whether offood origin or as a result of nutritional supplement augmentation).Thus, the presence of the bifidogenic oligosaccharides in thecompositions of the present invention allows for more effectivemicrobial inhibition by increasing the ability of all varieties ofprobiotic bacteria to grow, and therefore provide said benefit.

The bifidogenic oligosaccharide of the present invention may be usedeither alone, or in combination with a lactic acid-producingmicroorganisms in a therapeutic composition. More specifically, due tothe growth promoting activity of bifidogenic oligosaccharides, thepresent invention contemplates a composition comprising a bifidogenicoligosaccharide present in a concentration sufficient to promote thegrowth of lactic acid-producing microorganisms. As shown herein, theseconcentrations amounts can vary widely, as the probiotic microorganismswill respond to any metabolic amount of nutrient oligosaccharide, andtherefore the present invention need not be so limited.

A preferred and exemplary bifidogenic oligosaccharide is FOS, althoughother carbohydrates may also be utilized, either alone or incombination. FOS can be obtained from a variety of natural sources,including commercial suppliers. As a product isolated from naturalsources, the components can vary widely and still provide the beneficialagent, namely FOS. FOS typically has a polymer chain length of fromabout 4 to 200 sugar units, with the longer lengths being preferred. Forexample, the degree of purity can Vary widely so long asbiologically-functional FOS is present in the final formulation.Preferred FOS formulations contain at least 50% by weight offructo-oligosaccharides compared to simple (mono or disaccharide) sugarssuch as glucose, fructose or sucrose, preferably at least 80%fructo-oligosaccharides (FOS), more preferably at least 90% and mostpreferably at least 95% FOS. Sugar content and composition can bedetermined by any of a variety of complex carbohydrate analyticaldetection methods as is well known. Preferred sources of FOS include,but are not limited to: inulin; Frutafit IQ™ (Imperial Suiker Unie;Sugar Land, Tex.); NutraFlora™ (Americal Ingredients, Inc.; Anaheim,Calif.); and Fruittrimfat Replacers and Sweeteners (Emeryville, Calif.).Bifidogenic oligosaccharides such as GOS, and other long chainoligosaccharides are also available from commercial vendors.

5. Diatomaceous Earth

Diatomaceous earth is the skeletal remains of single cell aquatic plantsknown as diatoms which are typically relatively uniform in composition,depending upon the source of the deposit and the component species ofdiatoms present in the deposit. Diatomaceous earth is characterized ashaving a silica content, a characteristic morphological shape, dependingupon the species, and an average size of from about 5 to 20 microns (μm)in diameter.

Different species of diatoms in diatomaceous earth provide a diverserange of shapes, providing different degrees of sharp and/or spiny edgeswhich when contacted with insects, parasites and small microorganismspierce the protective coatings of the target parasite/pathogen.Diatomaceous earth is included in a therapeutic composition of thisinvention in a wide variety of concentrations, depending upon the mannerof administration. Typical compositions contain from about 0.1 to 99%weight of diatomaceous earth per weight (w/w) of composition. Forconcentrated single dose uses, a high content of diatomaceous earth isused, typically 5 to 50% w/w, and preferably about 5 to 10% w/w. Forcontinuous feed applications, a moderate to low content of diatomaceousearth is used, typically 0.5 to 10% w/w, and preferably 1 to 5% w/w.

A preferred diatomaceous earth for use in a composition of the presentinvention has a low ash content, typically less than 1% w/w, a lowamorphous silica content, typically less than 1% w/w, and a lowvolconoclastic sediment, typically less than 1% w/w. Insofar as apreferred diatomaceous earth has the further property of presentingsharp and/or spiny edges to damage the external protective surfaces ofthe parasite/pathogen to be inhibited. Diatom shapes are wellcharacterized in art, and the spiny, sharp character can be easilyobserved by microscopic examination of the diatoms. By observation andquantitative analysis, one can readily determine the proportions of thecomponent diatoms in the diatomaceous earth. A preferred diatomaceousearth contains a high content of abrasive diatoms. A particularlypreferred diatomaceous earth contains Melosira diatoms, and preferablyis comprised of at least 50% w/w Melosira diatoms, more preferably atleast 70% w/w Melosira diatoms, and most preferably at least 80%Melosira diatoms.

Diatomaceous earth can be obtained from a variety of sources. Typically,any diatom deposit is a source of diatomaceous earth. Commercialsuppliers routinely mine, characterize and provide different grades ofdiatomaceous earth. A particularly preferred supplier of diatomaceousearth rich in Melosira diatoms is the CR Minerals Corporation, Golden,Colo.

6. Methods of Producing or Enhancing Antibiotic Resistance

As previously discussed, the present invention discloses methodologiesfor the selection, isolation, and culturing of antibiotic-resistantstrains of lactic acid-producing bacteria be used as concomitantlyadministered biorational agents. These embodiments may be predicatedupon:

(i) selectively culturing the probiotic bacteria (which may initially besensitive to the antibiotic of choice) in gradually increasingconcentrations of antibiotic of choice in order to facilitate thedevelopment of decreased antibiotic sensitivity or, preferably,antibiotic resistance; (ii) utilizing “conjugative transposons” (i.e.,DNA sequences that allow the direct transfer of resistance genes withouta plasmid intermediary) to confer antibiotic resistance to an antibioticsensitive bacterial stain; and (iii) utilizing plasmids (i.e., small,non-chromosomal, autonomously replicating, circular DNA which oftennaturally possess antibiotic resistance genes) possessing genesconferring resistance to the antibiotic of choice which are generated bystandard Recombinant DNA-based techniques.

It should be noted, however, that the most preferred embodiment of thepresent invention utilizes the selective culturing of the probioticbacteria in gradually increasing concentrations of antibiotic of choicein order to facilitate the development of decreased antibioticsensitivity or, preferably, antibiotic resistance. This embodiment,which will be more fully discussed below, is preferred due to the factthat current FDA and other governmental agency regulations expresslyprohibit the intentional release of “man-made” (e.g., recombinant)antibiotic resistant bacterial strains into the environment. Hence, theutilization of the antibiotic resistant strains of bacteria disclosed inthe present invention, produced through the non-recombinant-based,selective culture-based methodology, would not be violative of theseaforementioned regulations. It should be noted, however, that thepreference of this embodiment is not intended to be limiting, but ratherreflects current regulations governing this field of endeavor. Shouldthese regulations be modified, or if new regulations are promulgated,the inventors fully intend to utilize all methodologies disclosed hereinto practice the present invention in the most efficacious mannerpossible.

Bacillus coagulans (strain ATCC Accession No. 31284) was assayed forantibiotic resistance/sensitivity utilizing the Kirby-Bauer agardilution method (see e.g., Bergey's Manual of Systemic Bacteriology,Vol. 2, Sneath, P. H. A. et al., eds., Williams & Wilkins, Baltimore,Md., 1986). This methodology demonstrated that this Bacillus coagulansstrain was susceptible to Piperacillin, Trimethoprim-Sulfamethoxasole,Ampicillin, Ciprofloxacin, Erythromycin, Vancomycin, Gentamicin, andOxacillin and was intermediate with respect to Clindamycin andTetracycline. Specifically, by Vitek, the MICs were found to be: (i)Ampicillin—2; (ii) Penicillin G—0.12; (iii) Vancomycin—<0.5; (iv)Nitrofurantoin—<32; (v) Norfioxacin—<4; (vi) Chloramphenicol—8; (vii)Clindamycin—>8 (resistant); and Tetracycline—>16 (resistant). Bacilluscoagulans (strain ATCC Accession No. 31284) possesses natural resistanceto the antibiotics Clindamycin and Tetracycline.

Subsequently, each prospective microorganism was then screened,utilizing the aforementioned methodology, for antibiotic sensitivity.Media and agars which were specific for each prospective bacteria weremixed with sub-lethal levels of the desired antibiotic compound. Forexample, DIFCO Trypticase Soy Agar (TSA) containing a sub-lethal levelof Vancomycin was prepared. The media/antibiotic mixture was thensterilized by steam autoclaving, ethylene oxide, or ionizing radiation(i.e., Gamma Processing) in cases where the antibiotic in question wassensitive to extreme heat. Petri dishes containing the agar/antibioticmixture were poured and the prospective microorganisms (selected from asingle colonies) were streaked on these plates.

Surviving (i.e., viable) bacterial colonies were then selected andtransferred to new antibiotic-containing media in which theconcentration of the selected antibiotic was gradually increased totherapeutic levels. At each stage of the selection process, survivingcolonies of Bacillus coagulans were selected and transferred to newmedia until therapeutic level antibiotic resistance is established.

7. Probiotic Activity of Bacillus coagulans

It is well-documented clinically that many species of bacterial, mycoticand yeast pathogens possess the ability to cause a variety ofgastrointestinal disorders including, but not limited to: disruption ofnormal gastrointestinal biochemical function, necrosis ofgastrointestinal tissues, and disruption of the bioabsorption ofnutrients, and like conditions. Therefore, the utilization of theprobiotic microorganism-containing compositions of the present inventioninhibits these pathogens are useful in the prophylactic or therapeutictreatment of conditions associated with infection by theseaforementioned pathogens.

7.1 Anti-Microbial Probiotic Activity

The ability of Bacillus coagulans to inhibit various bacterial pathogenswas quantitatively ascertained by use of an in vitro assay. This assayis part of a standardized bacterial pathogen screen (developed by theU.S. Food and Drug Administration (FDA)) and is commercially availableon solid support disks (DIFCO® BACTROL® Antibiotic Disks). To performthe assay, potato-dextrose plates (DIFCO®) were initially prepared usingstandard procedures. The plates were then individually inoculated withthe bacteria (approximately 1.5×10⁶ CFU) to be tested so as to form aconfluent bacterial bed.

Inhibition by Bacillus coagulans was subsequently ascertained by placingapproximately 1.8×10⁶ CFU of Bacillus coagulans in 10 μl of broth orbuffer, directly in the center of the potato-dextrose plate with onetest locus being approximately 8 mm in diameter per plate. A minimum ofthree test loci were used for each assay. The negative control consistedof a 10 μl volume of a sterile saline solution, whereas the positivecontrol consisted of a 1 μl volume of glutaraldehyde. The plates werethen incubated for approximately about 18 hr at 30° C., at which timethe zones of inhibition were measured. As designated herein, “excellentinhibition” means the zone was 10 mm or greater in diameter; and “goodinhibition” means the zone was greater than 2 mm in diameter but lessthan 10 mm in diameter.

As expected, no “inhibition” was seen with the negative, saline control,and excellent “inhibition” (approximately 16.2 mm diameter; average ofthree tests) was seen with the positive, glutaraldehyde control. For theenteric microorganisms tested, the following inhibition by Bacilluscoagulans was found: (i) Clostridium species—excellent inhibition; (ii)Escherichia coli—excellent inhibition; (iii) Clostridiumspecies—excellent inhibition, where the zone of inhibition wasconsistently greater than 15 mm in diameter. Similarly, excellentinhibition was also seen for the opportunistic pathogens Pseudomonasaeruginosa, and Staphylococcus aureus. In summation, pathogenic entericbacteria which were shown to be inhibited by Bacillus coagulans activityinclude, but are not limited to: Staphylococcus aureus; Staphylococcusepidermidis; Streptococcus pyogenes; Pseudomonas aeruginosa; Escherichiacoli (enterohemorragic species); numerous Clostridium species (e.g.,Clostridium perfingens, Clostridium botulinum, Clostridium tributrycum,Clostridium sporogenes, and the like); Gardnereia vaginails;Proponbacterium aenes; Aeromonas hydrophia; Aspergillus species; Proteusspecies; and Klebsiella species.

7.2 Anti-Mycotic Probiotic Activity

The ability of Bacillus coagulans to inhibit various fungal pathogenswas demonstrated using an in vitro assay. The tested fungal strains ofTrichophyton species are available from the American Type CultureCollection (ATCC; Rockville, Md.) and their ATCC accession numbers areillustrated in FIG. 2.

In the assay, potato-dextrose plates (DIFCO®, Detroit, Mich.) wereprepared using standard procedures and were inoculated individually witha confluent bed (about 1.7×10⁶) of various species of the fungusTrichophyton. Inhibition by Bacillus coagulans was ascertained byplacing on the plate approximately 1.5×10⁶ colony forming units (CFU) in10 μl of broth or buffer, plated directly in the center of thepotato-dextrose plate, with one test locus per plate. The size of eachtest locus was approximately 8 mm in diameter and a minimum of threetests were performed for each inhibition assay. The negative controlconsisted of a 10 ml volume of sterile saline solution, whereas thepositive control consisted of a 10 ml volume 2% Miconazole(1-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxylmethyl-1,11-imidazolewithin an inert cream.

The plates were then incubated for approximately 18 hr at 30° C., atwhich time the zones of inhibition were measured. As designated herein,“excellent inhibition” means the zone was 10 mm or greater in diameter;and “good inhibition” means the zone was greater than 2 mm in diameter,but less than 10 mm in diameter.

The results of in vitro inhibition by Bacillus coagulans are illustratedin FIG. 2. For each of the Trichophyton species tested, the diseasecondition associated with an infection is indicated in column 2 of FIG.2. For comparison, no zone of inhibition was seen with the negativecontrol, whereas good inhibition (approximately 8.5 mm diameter, meanaverage of three tests) was seen with the positive control.

7.3 Probiotic Inhibition of Yeast

Similarly, the ability of Bacillus coagulans to inhibit various yeastpathogens was demonstrated in vitro for four species of Candida, all ofwhich are available from the American Type Culture Collection (ATCC;Rockville, Md.) with their ATCC accession numbers illustrated in FIG. 3.

In the assay, potato-dextrose plates (DIFCO®, Detroit, Mich.) wereprepared using standard procedures and were inoculated individually witha confluent bed about 1.7×10⁶ of the four species of Candida. Inhibitionby B. coagulans was tested by placing on the plate about 1.5×10⁶ colonyforming units (CFU) in 10 μl of broth or buffer, plated directly in thecenter of the potato-dextrose plate with one test locus of about 8 mm indiameter per plate. A minimum of three tests were performed for eachinhibition assay. The negative control consisted of a 1 ml volume of asterile saline solution, whereas the positive control consisted of a 1ml volume of Miconazole cream.

The plates were then incubated for approximately 18 hr at 30° C., atwhich time the zones of inhibition were measured. As designated herein,“excellent inhibition” means the zone was 10 mm or greater in diameter;and “good inhibition” means the zone was greater than 2 mm in diameter,but less than 10 mm in diameter.

The results of the in vitro tests are shown in FIG. 3 with thepathological conditions in humans associated with infection by theCandida species shown in column 2. As expected, no inhibition was seenwith the negative control and good inhibition (approximately 8.7 mmdiameter; average of three tests) was seen with the positive control.

8. Therapeutic Compositions

8.1 Anti-Microbial Agent-Containing Therapeutic Compounds

It should be noted that although Bacillus coagulans is utilized hereinas a preferred exemplary probiotic species, by virtue of the commonphysiological characteristics which are indigenous to all lacticacid-producing bacteria, other species of these lactic acid-producingbacteria may be effectively in the methods and/or therapeuticcompositions disclosed in the present invention. Preferred, exemplaryformulations of the therapeutic compositions of the present inventionare set forth in FIG. 4.

The cells/spores can be presented in a variety of compositions suitedfor oral administration to the gastrointestinal tract, directed at theobjective of introducing the bacteria to tissues of the gastrointestinaltract. Therapeutic compositions of the present invention are, forexample, comprised of a lactic acid-producing bacteria strain,preferably vegetative Bacillus coagulans, Bacillus coagulans spores, orcombinations thereof which are a co-administrated with a selected agentswhich possesses the ability to ameliorate infections which have abacterial, fungal, and/or yeast etiology. In the aforementionedembodiment, the active lactic acid-producing bacteria species of thepresent invention comprise approximately 0.1% to 50% by weight of thefinal composition and, preferably, approximately 1% to 10% by weight,contained within a formulation suitable for oral administration. Morespecifically, the therapeutic composition of the present invention maycontain, within a 350 mg dosage formulation, for example, approximately2×10⁶ to 1×10¹⁰ colony forming units (CFU) of viable, lacticacid-producing vegetative bacteria or bacterial spores (in the case ofBacillus coagulans).

The formulation for a therapeutic composition of the present inventionmay also include other probiotic agents' or nutrients which promotespore germination and/or bacterial growth. A particularly preferredmaterial is a bifidogenic oligosaccharide, which promotes the growth ofbeneficial probiotic bacteria as previously described, supra.Bifidogenic oligosaccharides (e.g., fructo-oligosaccharide (FOS) orgluco-oligosaccharide (GOS)) may be utilized in various combinations,depending upon the specific formulation. The preferred therapeuticcomposition includes approximately 10 to 200 mg of bifidogenicoligosaccharide, and most preferably a concentration of approximately100 to 500 mg of bifidogenic oligosaccharide per unit of the therapeuticcomposition. Additionally, the therapeutic composition of the presentinvention may include other probiotic agents or nutrients for promotinggrowth, as well as other physiologically-active constituents which donot interfere with the overall therapeutic efficacy of the other activeagents contained within the therapeutic composition.

In another embodiment of the present invention, the Bacillus coagulansstrain is combined with a therapeutically-effective dose of an(preferably, broad-spectrum) antibiotic. The therapeutic composition ofthe present invention may also contain approximately 1 to approximately250 mg of the selected antibiotic per unit of therapeutic composition.In preferred embodiments of the present invention, the Bacilluscoagulans strain is combined with a therapeutic dose of an antibioticsuch as Gentamicin; Vancomycin; Oxacillin; Tetracyclines;Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-Sulfamethoxasole; a member of the Cephlosporin antibioticfamily (e.g., Cefaclor, Cefadroxil, Cefixime, Cefprozil, Ceftriaxone,Cefuroxime, Cephalexin, Loracarbef, and the like); a member of thePenicillin family of antibiotics (e.g., Ampicillin,Amoxicillin/Clavulanate, Bacampicillin, Cloxicillin, Penicillin VK, andthe like); with a member of the Fluoroquinolone family of antibiotics(e.g., Ciprofloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like); or amember of the Macrolide antibiotic family (e.g., Azithromycin,Erythromycin, and the like).

In another embodiment of the present invention, the Bacillus coagulansstrain is combined with a therapeutically-effective dose of ananti-fungal agent. The therapeutic composition of the present inventionmay also contain approximately 1 to 250 mg of the selected anti-fungalagent per unit of therapeutic composition. Typical anti-fungal agentswhich may be utilized include, but are not limited to: Clotrimazole,Fluconazole, Itraconazole, Ketoconazole, Miconazole, Nystatin,Terbinafine, Terconazole, Tioconazole, and the like.

In a preferred embodiment, Bacillus coagulans spores, atherapeutically-effective concentration of an antibiotic, anti-fungal,etc., and, if so desired, various other components (e.g., bifidogenicoligosaccharide, binders, etc.) are encapsulated into anenterically-coated, time-released capsule or tablet. The enteric coatingallows the capsule/tablet to remain intact (i.e., undissolved) as itpasses through the gastrointestinal tract, until such time as it reachesthe small intestine. Similarly, the time-released component prevents the“release” of the Bacillus coagulans spores for a pre-determined timeperiod which, preferably, will coincide with the end of the antibiotictreatment period as the antibiotic prevents the spores from geminatinguntil such time as the serum levels drop to a substantially low level.Once the antibiotic regimen is completed, the Bacillus coagulans sporesgerminate and this microorganism becomes the primary resident flora ofthe gastrointestinal tract, due to the killing-off of the previousresident flora by the antibiotic.

In addition, the vegetative Bacillus coagulans microorganisms do notadhere to the intestinal epithelium. Thus (without a repeat dosage), thebacteria remain in the gastrointestinal tract for maximal time ofapproximately 10 days and are considered to be a transient flora. Therelatively rapid gastrointestinal-clearance time and inability to adhereto the gastrointestinal epithelium of Bacillus coagulans, has theadvantage of preventing the later development of bacteremia in (forexample) immunocompromised individuals.

The therapeutic compositions of the present invention may also includeknown antioxidants, buffering agents, and other agents such as coloringagents, flavorings, vitamins or minerals. For example, a preferredtherapeutic composition may also contain one or more of the followingminerals: calcium citrate (15-350 mg); potassium gluconate (5-150 mg);magnesium citrate (5-15 mg); and chromium picollinate (5-200 μg). Inaddition, a variety of salts may be utilized, including calcium citrate,potassium gluconate, magnesium citrate and chromium picollinate.Thickening agents may be added to the compositions such aspolyvinylpyrrolidone, polyethylene glycol or carboxymethylcellulose.Preferred additional components of a therapeutic composition of thisinvention can include assorted colorings or flavorings, vitamins, fiber,enzymes and other nutrients. Preferred sources of fiber include any of avariety of sources of fiber including, but not limited to: psyllium,rice bran, oat bran, corn bran, wheat bran, fruit fiber and the like.Dietary or supplementary enzymes such as lactase, amylase, glucanase,catalase, and the like enzymes can also be included. Chemicals used inthe present compositions can be obtained from a variety of commercialsources, including Spectrum Quality Products, Inc (Gardena, Calif.),Sigma Chemicals (St. Louis, Mich.), Seltzer Chemicals, Inc., (Carlsbad,Calif.) and Jarchem Industries, Inc., (Newark, N.J.).

The various active agents (e.g., probiotic bacteria, antibiotics,anti-fungal agents, bifidogenic oligosaccharides, and the like) arecombined with a carrier which is physiologically compatible with thegastrointestinal tissue of the species to which it is administered.Carriers can be comprised of solid-based, dry materials for formulationinto tablet, capsule or powdered form; or the carrier can be comprisedof liquid or gel-based materials for formulations into liquid or gelforms. The specific type of carrier, as well as the final formulationdepends, in part, upon the selected route(s) of administration.

The therapeutic composition of the present invention may also include avariety of carriers and/or binders. A preferred carrier ismicro-crystalline cellulose (MCC) added in an amount sufficient tocomplete the one gram dosage total weight. Particularly preferredformulations for a therapeutic composition of this invention will bedescribed, infra. Carriers can be solid-based dry materials forformulations in tablet, capsule or powdered form, and can be liquid orgel-based materials for formulations in liquid or gel forms, which formsdepend, in part, upon the routes of administration.

Typical carriers for dry formulations include, but are not limited to:trehalose, maltodextrin, rice flour, micro-crystalline cellulose (MCC)magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and likecarriers. Where the composition is dry and includes evaporated oils thatproduce a tendency for the composition to cake (adherence of thecomponent spores, salts, powders and oils), it is preferred to includedry fillers which distribute the components and prevent caking.Exemplary anti-caking agents include MCC, talc, diatomaceous earth,amorphous silica and the like, and are typically added in an amount offrom approximately 1% to 95% by weight. It should also be noted that dryformulations which are subsequently rehydrated (e.g., liquid formula) orgiven in the dry state (e.g., chewable wafers, pellets or tablets) arepreferred to initially hydrated formulations. Dry formulations (e.g.,powders) may be added to supplement commercially available foods (e.g.,liquid formulas, strained foods, or drinking water supplies). Similarly,the specific type of formulation depends upon the route ofadministration.

Suitable liquid or gel-based carriers include but are not limited to:water and physiological salt solutions; urea; alcohols and derivatives(e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethyleneglycol, propylene glycol, and the like). Preferably, water-basedcarriers possess a neutral pH value (i.e., pH 7.0). The compositions mayalso include natural or synthetic flavorings and food-quality coloringagents, all of which must be compatible with maintaining viability ofthe lactic acid-producing microorganism. Well-known thickening agentsmay also be added to the compositions such as corn starch, guar gum,xanthan gum, and the like. Where a liquid-based composition containingspores is provided, it is desirable to include a spore germinationinhibitor to promote long term storage. Any spore germination inhibitormay be used. By way of example and not of limitation, preferredinhibitors include: hyper-saline carriers, methylparaben, guargum,polysorbates, preservatives, and the like.

Preservatives may also be included within the carrier includingmethylparaben, propylparaben, benzyl alcohol and ethylene diaminetetraacetate salts. Well-known flavorings and/or colorants may also beincluded within the carrier. The compositions of the present inventionmay also include a plasticizer such as glycerol or polyethylene glycol(with a preferred molecular weight of MW=800 to 20,000). The compositionof the carrier can be varied so long as it does not interferesignificantly with the pharmacological activity of the activeingredients or the viability of the Bacillus coagulans spores.

A composition can be formulated to be suitable for oral administrationin a variety of ways, for example in a liquid, a powdered foodsupplement, a paste, a gel, a solid food, a packaged food, a wafer, andthe like. Other formulations will be readily apparent to one skilled inthe art.

A nutrient supplement component of a composition of this invention caninclude any of a variety of nutritional agents, as are well known,including vitamins, minerals, essential and non-essential amino acids,carbohydrates, lipids, foodstuffs, dietary supplements, and the like.Preferred compositions comprise vitamins and/or minerals in anycombination. Vitamins for use in a composition of this invention caninclude vitamins B, C, D, E, folic acid, K, niacin, and like vitamins.The composition can contain any or a variety of vitamins as may bedeemed useful for a particularly application, and therefore, the vitamincontent is not to be construed as limiting. Typical vitamins are those,for example, recommended for daily consumption and in the recommendeddaily amount (RDA), although precise amounts can vary. The compositionwould preferably include a complex of the RDA vitamins, minerals andtrace minerals as well as those nutrients that have no established RDA,but have a beneficial role in healthy human or mammal physiology. Thepreferred mineral format would include those that are in either thegluconate or citrate form because these forms are more readilymetabolized by lactic acid bacteria. In a related embodiment, theinvention contemplates a composition comprising a viable lactic acidbacteria in combination with any material to be adsorbed, including butnot limited to nutrient supplements, foodstuffs, vitamins, minerals,medicines, therapeutic compositions, antibiotics, hormones, steroids,and the like compounds where it is desirable to insure efficient andhealthy absorption of materials from the gastrointestinal track into theblood. The amount of material included in the composition can varywidely depending upon the material and the intended purpose for itsabsorption, such that the invention is not to be considered as limiting.Other components of the compositions of the present invention can be abifidogenic oligosaccharide, as described herein.

By way of example, and not of limitation, Bacillus coagulans spores maybe incorporated into any type of dry or lyophilized product which isdissolved or mixed with hot water, so long as the temperature of theBacillus coagulans spore-containing mixture is raised to the requiredheat-shock temperature (i.e., 80° C. for 5 minutes) necessary forgermination of the spores. The Bacillus coagulans spores may either beincorporated into the dry or lyophilized product by the manufacturer ofthe product or by the consumer during preparation. These dry orlyophilized product include, but are not limited to: tea bags, coffee(e.g., “freeze-dried” or ground), sweeteners (e.g., synthetic(NutraSweet®) and natural); hot cereal (e.g., oatmeal, Cream of Wheat®,and the like), hot beverage condiments/flavorings and creamers, and thelike.

In another specific embodiment, Bacillus coagulans spores may beutilized as a dry or lyophilized product, or incorporated into achewable tablet, toothpaste, mouthwash, oral drops, and the like inorder to inhibit the formation of dental caries, gingivitis, and otherforms of periodontal disease. Similarly, Bacillus coagulans spores maybe incorporated, with or without anti-microbial agents, chewable tablet,toothpaste, mouthwash, oral drops, and the like in order to treat oralinfections caused by yeast (i.e., “thrush”), Herpes simplex I (i.e.,cold sores), and various other infections caused by oral pathogens.

In yet another specific embodiment, the Bacillus coagulans vegetativebacterial cells/spores may incorporated into an aqueous solution (e.g.,physiological saline) for administration as a colonic, via an enema orthe like) so as to directly administer the probiotic bacteria to thecolon. This method of administration is highly efficacious forutilization of vegetative bacterial cells as they are not exposed to thehighly acidic environment of the stomach as is the case during oraladministration.

8.2 Therapeutic Compositions Methods for Treating Bacterial Infections

The present invention contemplates a method for treating, reducing orcontrolling gastrointestinal bacterial infections using the therapeuticcomposition or therapeutic system disclosed herein. The disclosedmethods of treatment function so as to inhibit the growth of thepathogenic bacteria which are associated with gastrointestinalinfections, as well as to concomitantly mitigate the deleteriousphysiological effects/symptoms of these pathogenic infections.

Probiotic lactic acid bacterium, preferably Bacillus coagulans, aregenerally regarded as safe by those skilled within the art (i.e., GRASCertified by the FDA) and, therefore, suitable for direct ingestion infood stuffs or as a food supplement. The methods of the presentinvention comprise administration of a therapeutic compositioncontaining a viable lactic acid-producing bacteria to thegastrointestinal tract of a human or animal, to treat or preventbacterial infection. Administration is preferably made using a liquid,powder, solid food and the like formulation compatible with oraladministration, all formulated to contain a therapeutic composition ofthe present invention by use of methods well-known within the art.

The methods of the present invention includes administration of acomposition containing lactic acid-producing bacterial cells (i.e.,vegetative bacterial cells) and/or spores or isolated Bacillus coagulansextracellular products (which contains a metabolite possessingantibiotic-like properties) to a human or animal, so as to treat orprevent the colonization of antibiotic-resistant pathogens with thegastrointestinal tract. In particular, for VRE, VISA, PRP, and otherpathogens, the methods includes administering to the patient, forexample, Bacillus coagulans in food or as a food supplement. Oraladministration is preferably in an aqueous suspension, emulsion, powderor solid, either already formulated into a food, or as a compositionwhich is added to food by the user prior to consumption. Administrationto the gastrointestinal tract may also be in the form of an analsuppository (e.g., in a gel or semi-solid formulation). All suchformulations are made using standard methodologies.

Administration of a therapeutic composition is preferably to thegastrointestinal tract using a gel, suspension, aerosol spray, capsule,tablet, powder or semi-solid formulation (e.g., a suppository)containing a therapeutic composition of the present invention, allformulated using methods well-known within the art. Administration ofthe compositions containing the active probiotic lactic acid-producingbacterium which is effective in preventing or treating a pathogenicbacterial infection, generally consist of one to ten dosages ofapproximately 10 mg to 10 g of the therapeutic composition per dosage,for a time period ranging from one day to one month. Administrations are(generally) once every twelve hours and up to once every four hours. Inthe preferred embodiment, two to four administrations of the therapeuticcomposition per day, of approximately 0.1 g to 5 g per dose, for one toseven days. This preferred dose is sufficient to prevent or treat apathogenic bacterial infection. Of course, the specific route, dosageand timing of the administration will depend, in part, upon theparticular pathogen and/or condition being treated, as well as theextent of said condition.

A preferred embodiment of the present invention involves theadministration of from approximately 1×10³ to 1×10¹⁴ CFU of viable,vegetative bacteria or spore per day, more preferably from approximately1×10⁵ to 1×10¹⁰, and most preferably from approximately 5×10⁸ to 1×10⁹CFU of viable, vegetative bacteria or spores per day. Where thecondition to be treated involves antibiotic-resistant digestivepathogens and the patient is an adult, the typical dosage isapproximately 1×10² to 1×10¹⁴ CFU of viable, vegetative bacteria orspores per day, preferably from approximately 1×10⁸ to 1×10¹⁰, and morepreferably from approximately 2.5×10⁸ to 1×10¹⁰ CFU of viable,vegetative bacteria or spores per day. Where the condition to be treatedis Sudden Infant Death Syndrome (SIDS) and the patient is an infant over6 months old, the dosage is typically 1×10⁶ to 1×10⁹, preferably fromapproximately 5×10⁴ to 2.5×10⁵, and more preferably from approximately1.5×10⁵ to 2×10⁵ CFU of viable, vegetative bacteria or spores per day.

In addition, the present invention contemplates a method which comprisesoral administration of a composition that contains from approximately 10mg to 20 g of a bifidogenic oligosaccharide, preferably afructo-oligosaccharide (FOS), per day, preferably from approximately 50mg to 10 g, and more preferably from approximately 150 mg to 5 g perday, to preferentially promote the growth of the probiotic lacticacid-producing bacterium over the growth of the pathogen. The method canbe combined with treatment methods using a probiotic lacticacid-producing bacterium as described herein.

The present invention further contemplates a therapeutic system fortreating, reducing and/or controlling pathogenic bacterial infections.Typically, the system is in the form of a package containing atherapeutic composition of the present invention, or in combination withpackaging material. The packaging material includes a label orinstructions for use of the components of the package. The instructionsindicate the contemplated use of the packaged component as describedherein for the methods or compositions of the invention.

By way of example, and not of limitation, a system can comprise one ormore unit dosages of a therapeutic composition according to the presentinvention. Alternatively, the system can alternately contain bulkquantities of a therapeutic composition. The label contains instructionsfor using the therapeutic composition in either unit dose or in bulkforms as appropriate, and may also include information regarding storageof the composition, disease indications, dosages, routes and modes ofadministration and the like information.

Furthermore, depending upon the particular contemplated use, the systemmay optionally contain either combined or in separate packages one ormore of the following components: bifidogenic oligosaccharides,flavorings, carriers, and the like components. One particularlypreferred embodiment comprises unit dose packages of Bacillus spores foruse in combination with a conventional liquid product, together withinstructions for combining the probiotic with the formula for use in atherapeutic method.

9. Utilization of the Therapeutic Compositions of the Present Inventionin the Treatment of Bacterial Gastroenteritis

Several microbial species have been quantitatively ascertained as theetiology for the vast majority of food-borne gastrointestinal infection(i.e., bacterial gastroenteritis), with Campylobacter jejuni-mediatedcampylobacteriosis being the most commonly reported (46%) cause ofbacterial gastroenteritis in the United States, followed in prevalenceby Salmonella typhimurium-mediated salmonellosis (28%); shigellosis(17%); and Escherichia coli O157 infection (5%). In addition, it isquite possible that various Salmonella and Shigella species mayeventually acquire antibiotic resistance (i.e., Metacillin orVancomycin) in that same manner in which Enterococci originally acquiredantibiotic resistance from Staphylococcus aureus.

Although the methodologies disclosed in the present invention areequally applicable to the therapeutic intervention of all forms ofbacterial gastroenteritis, by way of example and not of limitation, thefollowing discussion will be primarily limited to the utilization ofthese methodologies in the treatment of Campylobacter jejuni-mediatedbacterial gastroenteritis.

Campylobacter jejuni was first identified as a human gastrointestinaltract (i.e., diarrheal) pathogen in 1973. As previously stated, in 1996,46% of all laboratory-confirmed cases of bacterial gastroenteritisreported to the Centers for Disease Control and Prevention (CDC) werecaused by Campylobacter species. In the United States alone, anestimated 2.1 to 2.4 million cases of human campylobacteriosis occureach year. See e.g., Tauxe, R. V. Epidemiology of Campylobacter jejuniinfections in the United States and other industrial nations. In:Campylobacter jejuni: current and future trends. P. 9-13 (Nachamkin, I.and Tompkins L. S., editors; American Society for Microbiology; 1992).Less frequently, Campylobacter jejuni infections have also been reportedto cause bacteremia, septic arthritis, and various otherextra-intestinal pathology. See e.g., Peterson, M. C., 1994. Wes. J.Med. 161: 148-152. In addition, an increasing proportion of humaninfections caused by Campylobacter jejuni are resistant toanti-microbial therapy. The mishandling of raw poultry and consumptionof undercooked poultry are the major risk factors for humancampylobacteriosis.

Deaths from Campylobacter jejuni-related infections are relatively rare,and occur primarily in infants, the elderly, and individuals withunderlying illnesses. For example, the incidence of campylobacteriosisin HIV-positive/AIDS patients is markedly higher than in the generalpopulation. In Los Angeles County between 1983 and 1987, the reportedincidence of campylobacteriosis in patients with AIDS was 519 cases per100,000 population, which is 39-times higher than the rate in thegeneral population. See e.g., Sorvillo, F. J. et al., 1991. J. AcquiredImmune Defic. Syndr. Hum. Retrovirol. 4: 595-602. Common complicationsof campylobacteriosis in HIV-infected individuals include recurrentinfections with antimicrobial-resistant bacterial strains. See e.g.,Penman, D. J. et al., 1988. Ann. Intern. Med. 108: 540-546.

9.1 Pathophysiology of Campylobacter jejuni-Mediated Gastroenteritis

The pathophysiology of Campylobacter jejuni-mediated gastroenteritisinvolves both host- and pathogen-specific factors. Factors including,but not limited to, the overall health and age of the host (see e.g.,Tauxe, R. V. Epidemiology of Campylobacter jejuni infections in theUnited States and other industrial nations. In: Campylobacter jejuni:current and future trends. P. 9-13 (Nachamkin, I. and Tompkins L. S.,editors; American Society for Microbiology; 1992) and Campylobacterjejuni-specific humoral immunity from previous exposure (see e.g.,Blaser, M. J. et al., 1987. JAMA 257: 43-46) influence the clinicaloutcome following infection.

The ingestion of relatively low numbers of viable organisms issufficient to cause infection in healthy adults. For example, in onevolunteer study, Campylobacter jejuni infection was demonstrated tooccur after the ingestion of as few as 800 organisms, with the overallrates of infection increasing as a function of the ingested dose. Seee.g., Black, R. E. et al., 1988. J. Infect. Dis. 157: 472-479. Inaddition, the rates of infection appeared to increase when bacterialinocula were ingested in a suspension buffered to reduce gastricacidity. See e.g., Black, R. E. et al., 1988. J. Infect. Dis. 157:472-479. Similarly, both rates of Campylobacter jejuni infectivity andthe severity of accompanying disease appeared to be positively effectedby disturbances in the overall gastrointestinal “health” of the infectedindividual (e.g., secondary disease or infection precipitating loweredlevels of normal gastrointestinal flora, and the like. In accord, thesensitivity of Campylobacter jejuni to decreased pH (i.e., acidicenvironments) and competing bacterial species serves to illustrate thepotential efficacy of the utilization of the antibiotic resistant,lactic acid-producing probiotic bacteria (in combination with theappropriate antibiotic) disclosed in the present invention to mitigatethe in vivo growth of Campylobacter jejuni, and hence its rate ofinfectivity, by generating an inhospitable acidic, competitiveenvironment within the individual's gastrointestinal tract.

Many pathogen-specific virulence determinants may contribute to thepathogenesis of Campylobacter jejuni-mediated infection, but none has aquantitatively ascertained role. See e.g., Ketley, J. M., 1997.Microbiology 143: 5-21. Suspected determinants of pathogenicity include,but are not limited to: chemotaxis, motility, and flagella, all of whichare required for attachment and colonization of the gastrointestinalepithelium. See e.g., Ketley, J. M., 1997. Microbiology 143: 5-21. Oncecolonization occurs, other possible virulence determinants are ironacquisition, host cell invasion, toxin production, inflammation andactive secretion, and epithelial disruption with leakage of serosalfluid. See e.g., Ketley, J. M., 1997. Microbiology 143: 5-21.

9.2 Sequelae to Campylobacter jejuni Infection

Several chronic, extra-gastrointestinal sequelae are associated withCampylobacter jejuni-mediated bacterial gastroenteritis (e.g.,Guillain-Barré syndrome and Reiter syndrome—a reactive type ofarthropathy).

Guillain-Barré syndrome (GBS), a demyelating disorder resulting in acuteneuromuscular paralysis, is a serious sequelae of Campylobacterinfection. See e.g., Allos, B. M., 1997. J. Infect. Dis. 176: 5125-5128.It has been estimated that one case of GBS occurs for every 1,000 casesof campylobacteriosis and up to 40% of patients with the syndrome havedemonstrated evidence of recent Campylobacter infection. Approximately20% of patients with GBS are left with some disability, andapproximately 5% die despite recent advances in respiratory care.

Campylobacteriosis is also associated with Reiter syndrome, a reactivearthropathy. See e.g., Peterson, M. C., 1994. Scand. J. Rheumatol. 23:167-170. In approximately 1% of patients with campylobacteriosis, thesterile post-infection process occurs 7 to 10 days after onset ofdiarrhea. Multiple joints can be affected, particularly the knee joint.Pain and incapacitation can last for months or, in some cases, becomechronic.

Both GBS and Reiter syndrome are thought to be autoimmune responsesstimulated by infection. For example, many individuals with Reitersyndrome have been found to carry the HLA B27 antigenic marker. Seee.g., Peterson, M. C., 1994. Scand. J. Rheumatol. 23: 167-170.Unfortunately, the pathogenesis of GBS (see e.g., Shoenfeld, Y. et al.,1996. Int. Arch. Allergy Immunol. 109: 318-326) and Reiter syndrome isnot completely understood.

9.3 Anti-Microbial-Resistant Strains of Campylobacter jejuni

The increasing rate of human infections caused byanti-microbial-resistant strains of Campylobacter jejuni makes clinicalmanagement of cases of campylobacteriosis markedly more difficult. Seee.g., Murphy, G. S. et al., 1996. Clin. Infect. Dis. 22: 568-569;Piddock, L. V., 1995. Antimicrob. Agents Chemother. 36: 891-898. Aspreviously discussed, anti-microbial resistance can prolong illness andcompromise treatment of patients with bacteremia. Interestingly, therate of anti-microbial-resistant Campylobacter jejuni-mediated entericinfections is highest in the developing world, where the use ofanti-microbial drugs in humans and animals is relatively unrestricted.For example, a 1994 study found that most clinical isolates ofCampylobacter jejuni from United States troops in Thailand weredemonstrated to be resistant to the broad spectrum antibiotic,Ciprofloxacin. Similarly, approximately one-third of Campylobacterjejuni isolates from United States troops located in Hat Yai were alsofound to be resistant to Azithromycin. See e.g., Murphy, G. S. et al.,1996. Clin. Infect. Dis. 22: 568-569.

In the industrialized world, the emergence of Campylobacter jejunistrains which have been found to be resistant to the broad spectrumantibiotic Fluoroquinolone, is illustrative of the need for prudentanti-microbial use in food-animal production. See e.g., Piddock, L. V.,1995. Antimicrob. Agents Chemother. 36: 891-898. Experimental evidencedemonstrates that Fluoroquinolone-susceptible Campylobacter jejunireadily become drug-resistant in poultry when these drugs areadministered. See e.g., Jacobs-Reitsma, W. F. et al., The induction ofquinolone resistance in Campylobacter bacteria in broilers by quinolonetreatment. In: Campylobacter, Helicobacters, and related organisms.1996. (Newell, D. G., Ketley, J. M., and Feldman, R. A., editors. NewYork: Plenum Press) p. 307-11. After Fluoroquinolone use in poultry wasapproved in Europe, resistant Campylobacter jejuni strains were shown torapidly emerge in humans during the early 1990's. See e.g., Piddock, L.V., 1995. Antimicrob. Agents Chemother. 36: 891-898. Similarly, within 2years of the 1995 approval of Fluoroquinolone use for poultry in theUnited States, the number of domestically-acquired human cases ofCiprofloxacin-resistant campylobacteriosis doubled in Minnesota. In a1997 study conducted in Minnesota, (20%) of 60 Campylobacter jejuniisolates obtained from chicken purchased in grocery stores were found tobe Ciprofloxacin-resistant. See e.g., Smith, K. E. et al.,Fluoroquinolone-resistant Campylobacter isolated from humans and poultryin Minnesota. 1995. Program of the 1st International Conference onEmerging Infectious Diseases; Mar. 7-10, 1998. Centers for DiseaseControl and Prevention; Atlanta, Ga.

9.4 Treatment of Campylobacter jejuni-Mediated Infections

Current, traditional therapeutic modalities primarily involve supportivemeasures, particularly fluid and electrolyte replacement, for mostpatients with campylobacteriosis. See e.g., Blaser, M. J., CampylobacterSpecies. In: Principles and practice of infectious diseases. 1990. p.1649-1658 (Mandell, G. L., ed., Churchhill Livingstone). Severelydehydrated patients should receive rapid volume expansion withintravenous fluids, however for most other patients, oral rehydration isindicated.

Although Campylobacter infections are generally self-limiting in nature,antibiotic therapy may be prudent for patients who have high fever,bloody diarrhea, or more than eight stools in 24 hours; immunosuppressedpatients, patients with systemic infections, and those whose symptomsworsen or persist for more than 1 week from the time of initialdiagnosis. When indicated, anti-microbial therapy soon after the onsetof symptoms can reduce the median duration of illness from approximately10 days to 5 days. However, when such treatment is delayed (e.g., untilCampylobacter jejuni infection is confirmed by a medical laboratory),antibiotic therapy may not be successful. Ease of administration, lackof serious toxicity, and high degree of efficacy make erythromycin thedrug of choice for Campylobacter jejuni infection; however, otheranti-microbial agents, particularly the quinolones and newer Macrolides(e.g., Azithromycin) may also utilized.

The utilization of antibiotic agents, which kill the “normal” microbialflora, frequently exacerbates the deleterious physiological effects(e.g., diarrhea, loss of the gastrointestinal mucosa, dehydration, andthe like) in individuals with Campylobacter jejuni-mediated bacterialgastroenteritis. Accordingly, the concomitant administration of anantibiotic and an antibiotic resistant probiotic microorganisms of thepresent invention to these individuals may ameliorate theseaforementioned deleterious physiological symptomology by re-establishingthe gastrointestinal microbial flora which serves to both directlycompete with the pathogenic bacteria for required growth moieties (e.g.,lipids, carbohydrates, electrolytes, amino acids, and the like), as wellas making the gastrointestinal environment inhospitable to the continuedgrowth of the pathogenic bacteria by lowering the pH through theproduction of lactic acid.

9.5 Other Bacterial Gastrointestinal Pathogens

Various other gastrointestinal pathogens, some antibiotic resistant,have been recently reported. These pathogens are amenable for preventionor treatment with the present invention.

For example, the FDA is investigating whether bacteria resistant toquinolone antibiotics can emerge in food animals and cause disease inhumans. Although thorough cooking has been demonstrated to sharplyreduce the likelihood of antibiotic-resistant bacteria surviving in meatinfect a human, pathogens resistant to drugs other than fluoroquinoloneshave been sporadically reported to survive in meat and subsequentlyinfect a human. In 1983, for example, 18 people in four midwesternstates developed multi-drug-resistant Salmonella food poisoning aftereating beef from cows fed antibiotics. Eleven of the people werehospitalized, and one died.

A study conducted by Cometta, et al., showed that increase in antibioticresistance parallels increase in antibiotic use in humans. See e.g.,Cometta, et al., 1994. New Engl. J. Med. 126: 43-47. They examined alarge group of cancer patients given fluoroquinolone antibiotics. Thepatients' white blood cell counts were very low as a result of theircancer treatment, thus leaving them open to opportunistic infection.Between 1983 and 1993, the percentage of such patients receivingantibiotics rose from 1.4 to 45. During those years, the researchersisolated Escherichia coli bacteria annually from the patients, andtested the microbes for resistance to five types of fluoroquinolones.Between 1983 and 1990, all 92 E. coli strains tested were easily killedby the antibiotics. But from 1991 to 1993, 11 of 40 tested strains (28percent) were found to be resistant to all five drugs.

10. Therapeutic Methods for Inhibiting Parasites in Animals

The present invention is also directed at methods for inhibiting growthof parasites and/or pathogenic organisms in the gastrointestinal tractof animals. The method comprises administering a composition of thepresent invention to the gastrointestinal tract of the animal, andthereby contact any parasites therein with an effective amount of theactive ingredients in the composition.

As used herein, the terms “pathogen” and “parasite” are usedinterchangeably in the context of a deleterious organism growing in thegastrointestinal tract and/or feces of an animal, although itappreciated that these terms have distinctive meanings.

The present invention describes methods for inhibiting growth of aparasite in the gastrointestinal tract of an animal comprising the stepof administering a composition of the invention to the gastrointestinaltract of the animal. A composition preferably contains diatomaceousearth and viable lactic acid-producing bacteria. The metabolic effect ofdiatomaceous earth present in a composition of this invention onparasites is to rupture tissues of the parasite, typically the softcuticle portions of the ectoskeleton, based on the abrasive quality ofthe diatomaceous earth upon cuticles arising during the mechanicaleffects of movement of the parasite after contacting the CE. Theseruptures in the cuticle breach the protective ectoskeleton of theparasite, rendering the parasite susceptible to infection, todehydration, to fluid exchanges and/or fluid losses, and the likeeffects which inhibit parasite health, and thereby inhibit growth.

The combined use of diatomaceous earth with an lactic acid-producingbacteria provides an beneficial synergy which provides importantbenefits to the claimed compositions, methods and systems. As describedherein, the use of the probiotic bacterial promotes healthy growth inthe intestinal tract, competing out deleterious bacteria, making thetissues targeted by the deleterious bacteria more healthy. Parasitescause local tissue damage at the site of growth and feeding, and oftenprovide inflammation and tissue injuries at the site as well. Thistissue damage provides a pathogenic or unhealthy environment where thetissue is ruptured and/or compromised in health, allowing undesirable oropportunistic pathogens to grow in the tissue vicinity. Because theparasite damages tissue and creates an environment that favorspathogenic infections, diatomaceous earth inhibits both the parasite andthe pathogenic infection by reducing the degree of tissue damage.Because the health of the host contributes to the ability to fight offthe parasite, improvements in tissue health by decreasing pathogenicinfections with probiotics increase the ability to inhibit parasitegrowth. Thus, the probiotic and the diatomaceous earth cooperate atinhibiting pathogens and parasites, respectively, which growth in turnpromotes growth of each other, decreasing tissue damage and increasingdigestive health of the host.

In one embodiment the invention contemplates methods for inhibitinggrowth of parasites and pathogenic organisms in the feces of animals.The method comprises administering a composition of the presentinvention into the gastrointestinal tract of an animal, therebyintroducing the active ingredients of the composition into theintestinal tract of the animal. A composition containing diatomaceousearth in an effective amount controls and/or inhibits parasite orpathogen growth in feces by first interfering with viable growth in theintestines, where the parasite first grows, thereby reducing the amountof parasite arriving in (i.e., “inoculating”) the feces, andsubsequently by interfering with growth that occurs in the feces afterthe feces is deposited.

Insofar as feces provide growth and breeding grounds for undesirableorganisms, controlling and/or inhibiting growth of parasites andpathogenic organisms in feces inhibits growth and reproduction of theseundesirable organisms in areas where feces is produced, deposited and/orstored. For example, in barns or corrals, in animal cages, in feed lots,in zoological display enclosures, and the like areas where animals aremaintained and feces is deposited, there is an opportunity forparasites/pathogens to irritate, spread, reproduce and/or infect otherhosts. These circumstances provide a variety of undesirable problemssolved by the present invention. For example, it is undesirable forparasites or pathogens to spread and further infect hosts, and thereofor any means to control spread of infection is of great benefit wheremultiple animals are caged together. In addition, in many circumstancesbiting of host animals by parasites or flying insects irritates and/orupsets animals, providing behavior problems which includes excessivekicking, biting and related activities which are unsafe for neighboringanimals and for animal handlers.

In a particularly preferred embodiment, the invention contemplates amethod for reducing and/or controlling flying insect populations inanimal cages/pens/enclosures where animals are maintained comprisingadministering a composition of the present invention to thegastrointestinal tract of the caged animals.

The present invention is useful at controlling a large variety ofparasites and pathogenic organisms, and therefore the invention need notbe limited to inhibiting any particular genus or species of organism.For example, based on the mechanisms described herein for effectivenessof the composition, it is seen that all insect varieties which can actas an animal parasite can be targeted by the methods of the presentinvention. Parasites can infect any of a variety of animals, includingmammals, reptiles, birds and the like, and therefore the invention isdeemed to not be limited to any particular animal. Examples ofwell-known or important parasites are described herein for illustrationof the invention, but are not to be viewed as limiting the invention.Representative parasites and animal and/or human hosts are described inextensive detail in a variety of veterinary treatises such as “Merck'sVeterinary Manual” and “Cecils' Human Diseases” Parasites of horsesincludes horse bots, lip bots or throat bots, caused by Gasterophilusspecies, such as G. intestinalis, G. haemorrhiodalis, and G. nasalis,stomach worms, caused by Habronema species, such as H. muscae or H.microstoma mulus, or caused by Crascia species, such as C. mepastoma, orcaused by Trichostrongylus species, such as T. axei, ascarids (whiteworms) caused by Parascaris species such as P. eciuorum, blood worms(palisade worms, red worms or sclerostomes) caused by Stroncrvlusspecies such as S. vulcraris, S. epuinus or S. edentatus, smallstrongyles of the cecum and colon caused by Triodontophorus species suchas T. tenuicollis, pinworms caused by Oxvuris species such as O. eaui,strongyloides infections of the intestine caused by Stroncivloideswesteri, tapeworms caused by Anonlocephala species such as A. macma andA. perfoliata, and caused by Paranonlocephala mamillana.

Various other parasites cause disease in ruminants, typically cattle,include the wire worm (or barber's pole worm or large stomach worm)caused by Haemonchus species. Parasites caused in ruminants, typicallyswine, include stomach worms caused by Hvostroncmulus species.

Additional parasites are known to infect a variety of animal hosts, andtherefore are a target for treatment by the methods of the presentinvention. For example, gastrointestinal parasites infect a variety ofanimals and can include Spirocerca species such as S. lupi that causeesopheageal worms in canines and Physoloptera species that cause stomachworms in canines and felines.

In humans, a large variety of parasites are particularly importanttargets for the methods of the present invention insofar as theseparasites are well known. However, the invention is not to be construedas limited to these parasites.

Where the animal is fed a pelletized or granular food, the compositioncan be included in the pelletized or granular food, or can comprise amixture of the pelletized food combined with a pelletized composition ofthis invention. Mixing pelletized food with a pelletized formulation ofa composition of this invention is a particularly preferred method forpracticing the present invention, insofar as it provides a convenientsystem for using commercial feeds and simultaneously regulating theamounts of a composition of this invention to be administered.

Administration of a therapeutic composition is preferably to the gutusing a gel, suspension, aerosol spray, capsule, tablet, granule,pellet, wafer, powder or semi-solid formulation (e.g., a suppository)containing a nutritional composition of this invention, all formulatedusing methods well known in the art.

The method comprises administration of a composition of this inventioncontaining the active ingredients to a human or animal in various dosageregimens as described herein to achieve the nutritional result.Administration of the compositions containing the active ingredientseffective in inhibiting parasite growth in the intestine and in fecesgenerally consist of one to ten unit dosages of 10 mg to 10 g per dosageof the composition for one day up to one month for an animal ofapproximately 100 kg body weight. Unit dosages are generally given onceevery twelve hours and up to once every four hours. Preferably two tofour dosages of the composition per day, each comprising about 0.1 g to50 g per dosage, for one to seven days are sufficient to achieve thedesired result.

A preferred method involves the administration into the digestive tractof from 1×10² to 1×10¹⁰ viable bacterium or spore per day, in someembodiments from 1×10³ to 1×10⁶, in other embodiments from 1×10⁶ to1×10⁹, and more preferably about from 5×10⁸ to 1×10⁹ viable bacterium orspore per day. Exemplary dosages range from about 1×10³ to 1×10⁶ viablebacterium per day, or alternatively range from about 1×10⁶ to 1×10⁹viable bacterium per day.

In a related embodiment, a preferred method comprises administration ofthe composition which delivers from about 0.1 to 25% weight ofdiatomaceous earth per volume (w/v) of composition, where thecomposition is typically formulated as an animal feed, preferably about0.5 to 10% (w/v), and more preferably about 1 to 5% (w/v). Typically,when used in animal feed a single dose route of administration will usea higher diatomaceous earth concentration, such as about 2 to 10% w/v,preferably about 5% w/v. When an animal feed route of administration isused in a daily feed mode, a lower diatomaceous earth concentration istypically used, for example about 0.5 to 2% w/v, preferably about 1%w/v. Stated differently, a typical unit dosage is a compositioncontaining about 50 milligrams (mg) to 10 grams of diatomaceous earth,preferably from about 200 to 500 mg, per 100 kilogram animal per day.

In addition, a preferred method comprises administering into thedigestive tract from 10 mg to 20 grams of fructo-oligosaccharide perday, preferably about 50 mg to 10 grams, and more preferably about from150 mg to 5 grams of fructo-oligosaccharide per day. These dosages areexpressed for an animal of approximately 70-100 kilogram body weight.For animals of other body sizes, the dosages are adjusted according tothe above body weight to dosage ratios.

The method is typically practiced on any animal where inhibitingpathogen or parasites is desired. The animal can be any livestock orzoological specimen where such inhibition of parasites/pathogensprovides economic and health benefits. Any animal can benefit by theclaimed methods, including birds, reptiles, mammals such as horses,cows, sheep, goats, pigs, and the like domesticated animals, or any of avariety of animals of zoological interest. Other purposes are readilyapparent to one skilled in the arts of nutrient absorption, feedutilization and bioavailability.

10.1. Therapeutic Systems for Inhibiting Parasite Growth

The present invention further contemplates a system for inhibitinggrowth of parasites and/or pathogens in the gastrointestinal tract of ananimal or in animal feces comprising a container comprising label and acomposition according to the present invention, wherein said labelcomprises instructions for use of the composition for inhibitingpathogen/parasite growth.

Typically, the system is present in the form of a package containing acomposition of this invention, or in combination with packagingmaterial. The packaging material includes a label or instructions foruse of the components of the package. The instructions indicate thecontemplated use of the package component as described herein for themethods or compositions of the invention.

For example, a system can comprise one or more unit dosages of atherapeutic composition according to the invention. Alternatively, thesystem can contain bulk quantities of a composition. The label containsinstructions for using the composition in either unit dose or in bulkforms as appropriate, and may include information regarding storage ofthe composition, feeding instruction, health and diet indications,dosages, routes of administration, methods for blending the compositionwith pre-selected food stuffs, and the like information.

EQUIVALENTS

From the foregoing detailed description of the specific embodiments ofthe present invention, it should be readily apparent that a uniquemethodology for the utilization of lactic acid-producing bacteria,preferably Bacillus coagulans, for the prevention and treatment ofgastrointestinal tract pathogens and their associated diseases, has beendescribed. Although particular embodiments have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims which follow. In particular, it iscontemplated by the inventor that various substitutions, alterations,and modifications may be made to the invention without departing fromthe spirit and scope of the invention as defined by the claims. Forinstance, the choice of the particular antibiotic which is utilized inthe Therapeutic Composition of the present invention is believed to be amatter of routine for a person of ordinary skill in the art withknowledge of the embodiments described herein.

1. A method for reducing gastrointestinal colonization by a pathogenicEscherichia coli bacterium, comprising identifying a mammalian subjecthaving an infection with said pathogenic Escherichia coli bacterium, andorally administering a therapeutically-effective concentration ofBacillus coagulans bacteria within a pharmaceutically-acceptable carriersuitable for administration to the gastrointestinal tract of saidsubject, wherein the Bacillus coagulans bacteria reduce colonization ofthe pathogenic Escherichia coli bacteria.
 2. The method of claim 1,further comprising the administration of a therapeutically-effectivedose of an antibiotic, wherein said antibiotic is selected from thegroup consisting of gentamicin, vancomycin, oxacillin, tetracycline,nitrofurantoin, chloramphenicol, clindamycin,trimethoprim-sulfamethoxasole, cefaclor, cefadroxil, cefixime,cefprozil, ceftriaxone, cefuroxime, cephalexin, loracarbef, ampicillin,amoxicillin/clavulanate, bacampicillin, cloxicillin, penicillin VK,ciprofloxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin,ofloxacin, sparfloxacin, trovafloxacin, azithromycin, and erythromycin.3. The method of claim 2, wherein the Bacillus coagulans bacteria areresistant to the antibiotic.
 4. The method of claim 1, furthercomprising the administration of a therapeutically-effective dose of anantibiotic, wherein the antibiotic is selected from the group consistingof a macrolide antibiotic, a cephalosporin antibiotic, a penicillinantibiotic, a fluoroquinolone antibiotic, and a vancomycin antibiotic.5. The method of claim 1, wherein the bacteria are in the form ofspores.
 6. The method of claim 1, wherein the Bacillus coagulans is B.coagulans Hammer strain deposited under ATCC accession number
 31284. 7.The method of claim 1, wherein approximately 2.5×10⁸ to approximately1×10¹⁰ viable Bacillus coagulans bacteria or spores are administered perday.
 8. The method of claim 1, wherein said mammalian subject is ahuman.
 9. The method of claim 4, wherein said Bacillus coagulansbacteria are resistant to said antibiotic.